Luminescent macrocyclic lanthanide complexes

ABSTRACT

The present invention provides a novel class of macrocyclic compounds as well as complexes formed between a metal (e.g., lanthanide) ion and the compounds of the invention. Preferred complexes exhibit high stability as well as high quantum yields of lanthanide ion luminescence in aqueous media without the need for secondary activating agents. Preferred compounds incorporate hydroxy-isophthalamide moieties within their macrocyclic structure and are characterized by surprisingly low, non-specific binding to a variety of polypeptides such as antibodies and proteins as well as high kinetic stability. These characteristics distinguish them from known, open-structured ligands.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/822,482 filed on Aug. 15, 2006, which is herein incorporatedby reference in its entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No.DE-AC03-76SF00098 awarded by the U.S. Department of Energy and GrantNos. AI063531 and EB004239 awarded by the National Institute of Health.The government has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to macrocyclic ligands and lanthanide complexesthereof, useful as luminescent markers, as well as methods utilizing theligands and complexes of the invention.

BACKGROUND OF THE INVENTION

There is a continuous and expanding need for rapid, highly specificmethods of detecting and quantifying chemical, biochemical andbiological substances as analytes in research and diagnostic mixtures.Of particular value are methods for measuring small quantities ofnucleic acids, peptides, pharmaceuticals, metabolites, microorganismsand other materials of diagnostic value. Examples of such materialsinclude small molecular bioactive materials (e.g., narcotics andpoisons, drugs administered for therapeutic purposes, hormones),pathogenic microorganisms and viruses, antibodies, and enzymes andnucleic acids, particularly those implicated in disease states.

The presence of a particular analyte can often be determined by bindingmethods that exploit the high degree of specificity which characterizemany biochemical and biological systems. Frequently used methods arebased on, for example, antigen-antibody systems, nucleic acidhybridization techniques, and protein-ligand systems. In these methods,the existence of a complex of diagnostic value is typically indicated bythe presence or absence of an observable “label” which has been attachedto one or more of the interacting materials. The specific labelingmethod chosen often dictates the usefulness and versatility of aparticular system for detecting an analyte of interest. Preferred labelsare inexpensive, safe, and capable of being attached efficiently to awide variety of chemical, biochemical, and biological materials withoutsignificantly altering the important binding characteristics of thosematerials. The label should give a highly characteristic signal, andshould be rarely, and preferably never, found in nature. The labelshould be stable and detectable in aqueous systems over periods of timeranging up to months. Detection of the label is preferably rapid,sensitive, and reproducible without the need for expensive, specializedfacilities or the need for special precautions to protect personnel.Quantification of the label is preferably relatively independent ofvariables such as temperature and the composition of the mixture to beassayed.

A wide variety of labels have been developed, each with particularadvantages and disadvantages. For example, radioactive labels are quiteversatile, and can be detected at very low concentrations. Such labelsare, however, expensive, hazardous, and their use requires sophisticatedequipment and trained personnel. Thus, there is wide interest innon-radioactive labels, particularly in labels that are observable byspectrophotometric, spin resonance and luminescence techniques, andreactive materials, such as enzymes that produce such molecules.

Labels that are detectable using fluorescence spectroscopy are ofparticular interest, because of the large number of such labels that areknown in the art. Moreover, the literature is replete with syntheses offluorescent labels that are derivatized to allow their facile attachmentto other molecules, and many such fluorescent labels are commerciallyavailable.

In addition to being directly detected, many fluorescent labels operateto quench the fluorescence of an adjacent second fluorescent label.Because of its dependence on the distance and the magnitude of theinteraction between the quencher and the fluorophore, the quenching of afluorescent species provides a sensitive probe of molecular conformationand binding, or other, interactions. An excellent example of the use offluorescent reporter quencher pairs is found in the detection andanalysis of nucleic acids.

Conventional organic fluorophores generally have short fluorescencelifetimes, on the order of nanoseconds (ns), which is generally tooshort for optimal discrimination from background fluorescence. Analternative detection scheme, which is theoretically more sensitive thanconventional fluorescence, is time-resolved luminescence. According tothis method, a chelated lanthanide metal with a long radiative lifetimeis attached to a molecule of interest. Pulsed excitation combined with agated detection system allows for effective discrimination againstshort-lived background emission. For example, using this approach, thedetection and quantification of DNA hybrids via an europium-labeledantibody has been demonstrated (Syvanen et al., Nucleic Acids Research14: 1017-1028 (1986)). In addition, biotinylated DNA was measured inmicrotiter wells using Eu-labeled streptavidin (Dahlen, Anal. Biochem.(1982), 164: 78-83). A disadvantage, however, of these types of assaysis that the label must be washed from the probe and its luminescencedeveloped in an enhancement solution.

In view of the predictable practical advantages it has been generallydesired that the lanthanide chelates employed should exhibit a delayedluminescence with decay times of more than 10 μs. The luminescence ofmany of the known luminescent chelates tends to be inhibited by water.As water is generally present in an assay, particularly an immunoassaysystem, lanthanide complexes that undergo inhibition of luminescence inthe presence of water are viewed as somewhat unfavorable or impracticalfor many applications. Moreover, the short luminescence decay times isconsidered a disadvantage of these compounds. This inhibition is due tothe affinity of the lanthanide ions for coordinating water molecules.When the lanthanide ion has coordinated water molecules, the absorbedlight energy (excitation energy) is quenched rather than being emittedas luminescence.

Thus, lanthanide chelates, particularly coordinatively saturatedchelates that exhibit excellent luminescence properties are highlydesirable. Alternatively, coordinatively unsaturated lanthanide chelatesexhibiting acceptable luminescence in the presence of water are alsoadvantageous. Such chelates that are derivatized to allow theirconjugation to one or more components of an assay find use in a range ofdifferent assay formats. The present invention provides these and othersuch compounds and assays using these compounds. Hydroxyisophthalamide(IAM) complexes of lanthanide ions such as Tb³⁺ are potentially usefulin a variety of biological applications. Of particular importance forbiological applications is that these complexes exhibit kineticstability in aqueous solutions at concentrations at or below nM levels.

Hydroxyisophthalamide ligands useful in applications requiringluminescence have been described (Petoud et al., J. Am. Chem. Soc. 2003,125, 13324-13325; U.S. Pat. No. 7,018,850 to Raymond et al.). The H(2,2)backbone has been employed to synthesize isophthalamide-based ligandssuch as 1 and 2 in FIG. 1. Those octadentate ligands display relativelyhigh thermodynamic stability when chelated to trivalent lanthanide ions.The functionalized TIAM ligand 2 has been conjugated to biomolecules andused as a donor in TR-LRET studies (Johansson et al., J. Am. Chem. Soc.2004, 126(50):16451-16455).

However, a need for luminescent complexes, which are stable underbiologically relevant conditions and at low concentrations, and whichsimultaneously exhibit low non-specific interactions with proteins,remains. The current invention addresses these and other needs.

SUMMARY OF THE INVENTION

This invention provides a new class of macrocyclic ligands and metalcomplexes thereof. In particular, the invention provides luminescentlanthanide complexes. Even more particularly, the invention providesluminescent terbium and europium complexes. These complexes exhibit highstability and solubility in aqueous media as well as high quantum yieldsof lanthanide ion luminescence in water without external augmentation,such as by micelles or fluoride. The complexes are formed between ametal ion of the lanthanide series and a new class of macrocyclicligands. Preferred ligands incorporate hydroxy-isophthalamide moietieswithin their structure and are characterized by surprisingly lownon-specific binding to a variety of different polypeptides such asantibodies and proteins. Due to their unique chemical andphysicochemical properties the complexes of the present invention couldfind use in any application requiring luminescence in aqueous media,including medical diagnostics and bioanalytical assay systems.

Thus, in a first aspect, the current invention provides a compoundhaving a structure according to Formula (I):

wherein the compound is covalently modified with at least one functionalmoiety.

In Formula I, each Z is a member independently selected from O and S.L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹ and L¹⁰ are linker groupsindependently selected from substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl and substituted orunsubstituted heterocycloalkyl.

A¹, A², A¹ and A⁴ are members independently selected from the followingstructure:

wherein each R¹ is a member independently selected from H, anenzymatically labile group, a hydrolytically labile group, ametabolically labile group and a single negative charge. Each R⁵, R⁶ andR⁷ is a member independently selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl, halogen,CN, CF₃, acyl, —SO₂NR¹⁷R¹⁸, —NR¹⁷R¹⁸, —OR¹⁷, —S(O)₂R¹⁷, —C(O)R¹⁸,—COOR¹⁷, —CONR¹⁷R¹⁸, —S(O)₂OR¹⁷, —OC(O)R¹⁷, —C(O)NR¹⁷R¹⁸, —NR¹⁷C(O)R¹⁸,—NR¹⁷SO₂R¹⁸ and —NO₂, wherein R⁶ and a member selected from R⁵, R⁷ andcombinations thereof are optionally joined to form a ring system whichis a member selected from substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl.

R¹⁷ and R¹⁸ are members independently selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl and substituted or unsubstituted heterocycloalkyl, and R¹⁷and R¹⁸, together with the atoms to which they are attached, areoptionally joined to form a 5- to 7-membered ring.

In a second aspect, the invention provides a luminescent complex formedbetween at least one metal ion and a compound of the invention.

In a third aspect, the invention provides a method of detecting thepresence or absence of an analyte in a sample. The method comprises (a)contacting the sample and a composition including a complex of theinvention; (b) exciting the complex; and (c) detecting luminescence fromthe complex. In one example, the presence or absence of the analyte isindicated by the absence or presence of luminescence from the complex.

In a fourth aspect, the invention provides a method of detecting thepresence or absence of an analyte in a sample. The method includes (a)contacting the sample and a composition including a complex of theinvention, and a luminescence modifying group, wherein energy can betransferred between the complex and the luminescence modifying groupwhen the complex is excited, and wherein the complex and theluminescence modifying group can be part of the same molecule or be partof different molecules; and (b) exciting said complex; and (c)determining the luminescent property of the sample, wherein the presenceor absence of the analyte is indicated by the luminescent property ofthe sample. In one example, the presence or absence of the analyte inthe sample is indicated by a change in the luminescent property of thesample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart indicating the kinetic stability of compound 1 undervarious conditions at room temperature.

FIG. 2 is a chart indicating the kinetic stability of compound 3 undervarious conditions at room temperature.

FIG. 3 is a chart indicating the kinetic stability of compound 5A undervarious conditions at room temperature.

FIG. 4 is a chart indicating that the emission spectrum of unconjugated5a-Tb is comparable to the emission spectra obtained for differentprotein conjugates of 5a-Tb.

FIG. 5 is a chart comparing the luminescence decay lifetimes ofunconjugated and streptavidin conjugated 5a-Tb.

FIG. 6 is a chart indicating steady-state absorption and emissionspectra which were recorded for compound 4-Tb.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

“Analyte”, as used herein, means any compound or molecule of interestfor which a diagnostic test is performed, such as a biopolymer or asmall molecular bioactive material. An analyte can be, for example, aprotein, peptide, carbohydrate, polysaccharide, glycoprotein, hormone,receptor, antigen, antibody, virus, substrate, metabolite, transitionstate analog, cofactor, inhibitor, drug, dye, nutrient, growth factor,lipid etc., without limitation.

As used herein, “energy transfer” refers to the process by which thelight emission of a luminescent group is altered by aluminescence-modifying group. When the luminescence-modifying group is aquenching group then the light emission from the luminescent group isattenuated (quenched). Energy transfer mechanisms include luminescenceresonance energy transfer by dipole-dipole interaction (e.g., in longerrange energy transfer) or electron transfer (e.g., across shorterdistances). While energy transfer is often based on spectral overlappingof the emission spectrum of the luminescent group and the absorptionspectrum of the luminescence-modifying group, (in addition to distancebetween the groups) it has been demonstrated that spectral overlap isnot necessarily required for energy transfer to occur (see e.g., Latvaet al., U.S. Pat. No. 5,998,146, which is incorporated herein byreference). It is to be understood that any reference to “energytransfer” herein encompasses all mechanistically-distinct phenomena.

“Energy transfer pair” is used to refer to a group of molecules thatparticipate in energy transfer. Such complexes may comprise, forexample, two luminescent groups, which may be different from one-anotherand one quenching group, two quenching groups and one luminescent group,or multiple luminescent groups and multiple quenching groups. In caseswhere there are multiple luminescent groups and/or multiple quenchinggroups, the individual groups may be different from one another.Typically, one of the molecules acts as a luminescent group, and anotheracts as a luminescence-modifying group. The preferred energy transferpair of the invention comprises a luminescent group and a quenchinggroup of the invention. There is no limitation on the identity of theindividual members of the energy transfer pair described herein. Allthat is required is that the spectroscopic properties of the energytransfer pair as a whole change in some measurable way if the distancebetween the individual members is altered by some critical amount.

As used herein, “luminescence-modifying group” refers to a molecule ofthe invention that can alter in any way the luminescence emission from aluminescent group. A luminescence-modifying group generally accomplishesthis through an energy transfer mechanism. Depending on the identity ofthe luminescence-modifying group, the luminescence emission can undergoa number of alterations, including, but not limited to, attenuation,complete quenching, enhancement, a shift in wavelength, a shift inpolarity, and a change in luminescence lifetime. One example of aluminescence-modifying group is a fluorescence-modifying group. Anotherexemplary luminescence-modifying group is a quenching group.

As used herein, “quenching group” refers to any luminescence-modifyinggroup of the invention that can attenuate at least partly the lightemitted by a luminescent group. This attenuation is referred to hereinas “quenching”. Hence, excitation of the luminescent group in thepresence of the quenching group leads to an emission signal that is lessintense than expected, or even completely absent. Quenching typicallyoccurs through energy transfer between the luminescent group and thequenching group.

“Fluorescence resonance energy transfer” or “FRET” is usedinterchangeably with “FET”, and “luminescence resonance energy transfer(LRET)” and refers to an energy transfer phenomenon in which the lightemitted by an excited luminescent group is absorbed at least partiallyby a luminescence-modifying group of the invention. Theluminescence-modifying group can, for instance, be a quenching group.LRET depends on energy transfer between the luminescent group and theluminescence-modifying group. LRET also depends on the distance betweenthe luminescence modifying group and the luminescent group.

“High dilution” or “H.D.” conditions as discussed herein refers toconditions which are better suited to obtain the desired product ratherthan undesired products. In performing chemical reactions especiallycyclization reactions, high dilution conditions are achieved by keepingthe concentration of one or more reactants low enough to reduce theformation of undesired polymeric by-products. Different reactions havedifferent reactant concentration requirements that can be worked out toobtain the desired yield of the desired product. One of ordinary skillin the art would be able to adjust the reactant concentrations in orderto achieve the desired yield of the desired product. In an exemplaryembodiment, the compounds of the invention that are prepared under highdilution conditions are prepared in such as way that the concentrationof at least one reactant is very low (approximately 1×10−5 M or less).Desired products can be obtained for reactions at higher concentrations,but the yield of desired products will be lower and the yields ofundesired products will be higher.

“Moiety” refers to the radical of a molecule that is attached to anothermoiety.

The term “targeting moiety” is intended to mean any moiety attached tothe complexes of the invention. The targeting moiety can be a smallmolecule, which is intended to include both non-peptides and peptides.The targeting group can also be a macromolecule, which includessaccharides, lectins, receptors, ligands for receptors, proteins such asBSA, antibodies, nucleic acids, solid supports and so forth. Thetargeting group can also be a lipid as well as a polymer, such as aplastic surface, a poly-ethyleneglycol derivative and the like.

As used herein, “nucleic acid” means DNA, RNA, single-stranded,double-stranded, or more highly aggregated hybridization motifs, and anychemical modifications thereof. Modifications include, but are notlimited to, those providing chemical groups that incorporate additionalcharge, polarizability, hydrogen bonding, electrostatic interaction, andfluxionality to the nucleic acid ligand bases or to the nucleic acidligand as a whole. Such modifications include, but are not limited to,peptide nucleic acids, phosphodiester group modifications (e.g.,phosphorothioates, methylphosphonates), 2′-position sugar modifications,5-position pyrimidine modifications, 8-position purine modifications,modifications at exocyclic amines, substitution of 4-thiouridine,substitution of 5-bromo or 5-iodo-uracil; backbone modifications,methylations, unusual base-pairing combinations such as the isobases,isocytidine and isoguanidine and the like. Modifications can alsoinclude 3′ and 5′ modifications such as capping with a SL, a fluorophoreor another moiety.

“Peptide” refers to a polymer in which the monomers are amino acids andare joined together through amide bonds, alternatively referred to as apolypeptide. When the amino acids are alpha-amino acids, either theL-optical isomer or the D-optical isomer can be used. Additionally,unnatural amino acids, for example, beta.-alanine, phenylglycine andhomoarginine are also included. Commonly encountered amino acids thatare not gene-encoded may also be used in the present invention. All ofthe amino acids used in the present invention may be either the D- orL-isomer. The L-isomers are generally preferred. The term “peptide” or“polypeptide”, as used herein, refers to naturally occurring as well assynthetic peptides. In addition, peptidomimetics are also useful in thepresent invention. For a general review, see, Spatola, A. F., inCHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES AND PROTEINS, B.Weinstein, eds., Marcel Dekker, New York, p. 267 (1983).

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below, such as “heteroalkyl.” Alkyl groups thatare limited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified, but notlimited, by —CH₂CH₂CH₂CH₂—, and further includes those groups describedbelow as “heteroalkylene.” Typically, an alkyl (or alkylene) group willhave from 1 to 24 carbon atoms, with those groups having 10 or fewercarbon atoms being preferred in the present invention. A “lower alkyl”or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N, S, B and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′— represents both —C(O)₂R′—and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, substituent that can be a single ring or multiple rings(preferably from 1 to 3 rings), which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) are meant to include both substituted and unsubstitutedforms of the indicated radical. Preferred substituents for each type ofradical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedheterocycloalkyl, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR″—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” The substituents are selected from, forexample: substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N),sulfur (S), silicon (Si) and boron (B).

The symbol “R” is a general abbreviation that represents a substituentgroup that is selected from substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, and substituted orunsubstituted heterocyclyl groups.

The present invention includes all salt forms of those molecules thatcontain ionizable functional groups, such as basic and acidic groups.The term “pharmaceutically acceptable salts” includes salts of theactive compounds which are prepared with relatively nontoxic acids orbases, depending on the particular substituents found on the compoundsdescribed herein. When compounds of the present invention containrelatively acidic functionalities, base addition salts can be obtainedby contacting the neutral form of such compounds with a sufficientamount of the desired base, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable base addition salts includesodium, potassium, calcium, ammonium, organic amino, or magnesium salt,or a similar salt. When compounds of the present invention containrelatively basic functionalities, acid addition salts can be obtained bycontacting the neutral form of such compounds with a sufficient amountof the desired acid, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable acid addition salts includethose derived from inorganic acids like hydrochloric, hydrobromic,nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge et al., Journal of Pharmaceutical Science,66: 1-19 (1977)). Certain specific compounds of the present inventioncontain both basic and acidic functionalities that allow the compoundsto be converted into either base or acid addition salts.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents, but otherwise the salts are equivalentto the parent form of the compound for the purposes of the presentinvention.

When a residue (such as R¹, R², R³ and R⁴) is defined herein as a singlenegative charge, then the residue can optionally include a cationiccounterion. The resulting salt form of the compound is encompassed inthe structure as presented.

In addition to salt forms, the present invention provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are encompassed within thescope of the present invention.

The graphic representations of racemic, ambiscalemic and scalemic orenantiomerically pure compounds used herein are taken from Maehr, J.Chem. Ed., 62: 114-120 (1985): solid and broken wedges are used todenote the absolute configuration of a chiral element; wavy linesindicate disavowal of any stereochemical implication which the bond itrepresents could generate; solid and broken bold lines are geometricdescriptors indicating the relative configuration shown but not implyingany absolute stereochemistry; and wedge outlines and dotted or brokenlines denote enantiomerically pure compounds of indeterminate absoluteconfiguration.

The terms “enantiomeric excess” and diastereomeric excess” are usedinterchangeably herein. Compounds with a single stereocenter arereferred to as being present in “enantiomeric excess,” those with atleast two stereocenters are referred to as being present in“diastereomeric excess.”

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(3H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

Introduction

The present invention provides a class of luminescent probes that arebased on metal chelates, which are formed between the metal ion and anovel class of macrocyclic ligands. In particular, the inventionprovides luminescent lanthanide complexes. Even more particularly, theinvention provides luminescent terbium and europium complexes. Thesecomplexes exhibit high stability as well as high quantum yields oflanthanide ion luminescence in aqueous media without the need forsecondary activating agents, such as by micelles or fluoride. Preferredligands incorporate hydroxy-phthalamide moieties within theirmacrocyclic structure and are characterized by surprisingly high kineticstability and unexpectedly low, non-specific binding to a variety ofdifferent polypeptides such as antibodies and proteins. Thesecharacteristics distinguish them from known, open-structured ligands.

The value of these lanthanide complexes derives from their high quantumefficiencies and relatively high absorption coefficients. Theseproperties make ligands such as compound 5 useful for homogeneous timeresolved luminescence resonance energy transfer (TR-LRET) applicationswhere donor and acceptor molecules are used at low concentrations.Complexes of the present invention could find use in any applicationrequiring strong luminescence under aqueous conditions including medicaldiagnostics and bioanalytical assay systems, such as immunoassays,peptide cleavage assays, DNA reporter assays and the like. In addition,these complexes and their derivatives may find wide applicability innanotechnology (incorporation into particles) and material science wherethe complexes could be embedded in solid materials that allow for thetransmission of light.

The fluorophores of the invention can be used with other fluorophores orquenchers as components of energy transfer probes. Many luminescent ornon-luminescent labels are useful in combination with the complexes ofthe invention and many such labels are available from commercialsources, such as SIGMA (Saint Louis) or Invitrogen, that are known tothose of skill in the art. Furthermore, those of skill in the art willrecognize how to select an appropriate fluorophore for a particularapplication and, if it is not readily available, will be able tosynthesize the necessary fluorophore or quencher de novo orsynthetically modify commercially available luminescent compounds toarrive at the desired luminescent label.

In addition to small-molecule fluorophores, naturally occurringfluorescent proteins and engineered analogues of such proteins areuseful with the compounds of the present invention. Such proteinsinclude, for example, green fluorescent proteins of cnidarians (Ward etal., Photochem. Photobiol. 1982, 35:803-808; Levine et al., Comp.Biochem. Physiol. 1982, 72B:77 85), yellow fluorescent protein fromVibrio fischeri strain (Baldwin et al., Biochemistry 1990, 29:5509 15),Peridinin-chlorophyll from the dinoflagellate Symbiodinium sp. (Morriset al., Plant Molecular Biology 1994, 24:673:77), phycobiliproteins frommarine cyanobacteria, such as Synechococcus, e.g., phycoerythrin andphycocyanin (Wilbanks et al., J. Biol. Chem. 1993, 268:1226 35), and thelike.

The compounds of the invention can be used as probes, as tools forseparating particular ions from other solutes, as probes in microscopy,enzymology, clinical chemistry, molecular biology and medicine. Thecompounds of the invention are also useful as therapeutic agents and asdiagnostic agents in imaging methods. Moreover, the compounds of theinvention are useful as components of optical amplifiers of light,waveguides and the like. Furthermore, the compounds of the invention canbe incorporated into inks and dyes, such as those used in the printingof currency and other instruments.

In one embodiment, the compounds of the invention show luminescenceafter exciting them in any manner known in the art, including, forexample, with light or electrochemical energy (see, for example, Kulmalaet al, Analytica Chimica Acta 1999, 386:1). The luminescence can, in thecase of chiral compounds of the invention, be circularly polarized (see,for example, Riehl et al., Chem. Rev. 1986, 86:1).

The compounds, probes and methods discussed in the following sectionsare generally representative of the compositions of the invention andthe methods in which such compositions can be used. The followingdiscussion is intended as illustrative of selected aspects andembodiments of the present invention and it should not be interpreted aslimiting the scope of the present invention.

Compositions

In a first aspect, the present invention provides a compound having astructure which is a member selected from:

In Formulae I and Ia, each Z is a member independently selected from Oand S. L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹ and L¹⁰ are linker groupsindependently selected from substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl and substituted orunsubstituted heterocycloalkyl. A¹, A², A³ and A⁴ are building blocks,which are members independently selected from substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl and a fused ring system.

In one embodiment, the compounds of Formulae I and Ia are covalentlymodified with a functional moiety. In an exemplary embodiment, at leastone of A¹, A², A³, A⁴, L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹ and L¹⁰ issubstituted with a functional moiety.

In another embodiment, the macrocyclic ligands of the invention arebased on hydroxy phthalic acid or hydroxy isophthalic acid orcombinations thereof as the building blocks. In an exemplary embodimentaccording to this aspect, A¹, A², A³ and A⁴ have a structure accordingto the following formula:

wherein each general structure for A¹, A², A³ and A⁴ is a memberindependently selected. Each R¹ is a member independently selected fromH, an enzymatically labile group, a hydrolytically labile group, ametabolically labile group and a single negative charge. Each R⁵, R⁶ andR⁷ is a member independently selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl, halogen,CN, CF₃, acyl, —SO₂NR¹⁷R¹⁸, —NR¹⁷R¹⁸, —OR¹⁷, —S(O)₂R¹⁷, —COOR¹⁷,—S(O)₂OR¹⁷, —OC(O)R¹⁷, —C(O)NR¹⁷R¹⁸, —NR¹⁷C(O)R¹⁸, —NR¹⁷SO₂R¹⁸, and—NO₂, wherein R⁶ and a member selected from R⁵, R⁷ and combinationsthereof are optionally joined to form a ring system, which is a memberselected from substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl andsubstituted or unsubstituted heteroaryl.

R¹⁷ and R¹⁸ are members independently selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl and substituted or unsubstituted heterocycloalkyl, and R¹⁷and R¹⁸, together with the atoms to which they are attached, areoptionally joined to form a 5- to 7-membered ring.

In another exemplary embodiment, the compound of the invention has thestructure:

wherein R¹, R², R³ and R⁴ are members independently selected from H, anenzymatically labile group, a hydrolytically labile group, ametabolically labile group and a single negative charge. Exemplarycompounds include those in which at least one of L¹, L², L³, L⁴, R⁵, R⁶,R⁷, R⁸, R⁹ R¹⁰, R¹¹, R¹², R¹⁴, R¹⁵ and R¹⁶ is substituted with afunctional moiety, and preferably at least one of L¹, L², L³, L⁴, L⁵,L⁶, L⁷, L⁸, L⁹ and L¹⁰ is substituted with a functional moiety.

R⁵, R⁶, R⁷, R⁸, R⁹ R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are membersindependently selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, halogen, CN, CF₃, acyl,—SO₂NR¹⁷R¹⁸, —NR¹⁷R¹⁸, —OR¹⁸; —S(O)₂R¹⁷, —COOR¹⁷, —S(O)₂OR¹⁷, —OC(O)R¹⁷,—C(O)NR¹⁷R¹⁸, —C(O)R¹⁸; —NR¹⁷C(O)R¹⁸, —NR¹⁷SO₂R¹⁸, and —NO₂, wherein R¹⁷and R¹⁸ are members independently selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl and substituted or unsubstituted heterocycloalkyl, whereinR¹⁷ and R¹⁸, together with the atoms to which they are attached, areoptionally joined to form a 5- to 7-membered ring. R⁶ and a memberselected from R⁵, R⁷ and combinations thereof are optionally joined toform a ring system. Likewise, R⁹ and a member selected from R⁸, R¹⁰ andcombinations thereof are optionally joined to form a ring system. Inaddition, R¹² and a member selected from R¹¹, R¹³ and combinationsthereof are optionally joined to form a ring system, wherein the ringsystem is a member selected from substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl and substituted or unsubstituted heteroaryl.

In an exemplary embodiment, in the compound of Formulae (I) or (Ia), L¹,L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹ and L¹⁰ are members independentlyselected from substituted or unsubstituted heteroalkylene andsubstituted or unsubstituted C₁ to C₆ alkylene. In an exemplaryembodiment, in the compound of Formulae (I) or (Ia), L¹, L², L⁴, L⁵ aremembers independently selected from substituted or unsubstitutedethylene. In an exemplary embodiment, in the compound of Formulae (I) or(Ia), L⁶, L⁷, L⁹, L¹⁰ are members independently selected fromsubstituted or unsubstituted ethylene. In an exemplary embodiment, inthe compound of Formulae (I) or (Ia), L⁶, L⁷, L⁹, L¹⁰ are membersindependently selected from substituted or unsubstituted methylene. Inan exemplary embodiment, in the compound of Formulae (I) or (Ia), L³ issubstituted or unsubstituted ethylene. In an exemplary embodiment, inthe compound of Formulae (I) or (Ia), L⁸ is substituted or unsubstitutedethylene. In an exemplary embodiment, in the compound of Formulae (I) or(Ia), L¹, L², L³, L⁴ and L⁵ are substituted or unsubstituted ethylene.In an exemplary embodiment, in the compound of Formulae (I) or (Ia), L⁶,L⁷, L⁸, L⁹, L¹⁰ are members independently selected from substituted orunsubstituted ethylene. In an exemplary embodiment, in the compound ofFormulae (I) or (Ia), L⁶, L⁷, L⁸, L⁹, L¹⁰ are members independentlyselected from substituted or unsubstituted methylene.

In an exemplary embodiment, in the compound of Formulae (II) or (IIa),L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹ and L¹⁰ are members independentlyselected from substituted or unsubstituted heteroalkylene andsubstituted or unsubstituted C₁ to C₆ alkylene. In an exemplaryembodiment, in the compound of Formulae (II) or (IIa), L¹, L², L⁴, L⁵are members independently selected from substituted or unsubstitutedethylene. In an exemplary embodiment, in the compound of Formulae (II)or (IIa), L⁶, L⁷, L⁹, L¹⁰ are members independently selected fromsubstituted or unsubstituted ethylene. In an exemplary embodiment, inthe compound of Formulae (II) or (IIa), L⁶, L⁷, L⁹, L¹⁰ are membersindependently selected from substituted or unsubstituted methylene. Inan exemplary embodiment, in the compound of Formulae (II) or (IIa), L³is substituted or unsubstituted ethylene. In an exemplary embodiment, inthe compound of Formulae (II) or (IIa), L⁸ is substituted orunsubstituted ethylene. In an exemplary embodiment, in the compound ofFormulae (II) or (IIa), L¹, L², L³, L⁴ and L⁵ are substituted orunsubstituted ethylene. In an exemplary embodiment, in the compound ofFormulae (II) or (IIa), L⁶, L⁷, L⁸, L⁹, L¹⁰ are members independentlyselected from substituted or unsubstituted ethylene. In an exemplaryembodiment, in the compound of Formulae (II) or (IIa), L⁶, L⁷, L⁸, L⁹,L¹⁰ are members independently selected from substituted or unsubstitutedmethylene.

In an exemplary embodiment, in the compound of Formulae (II) or (IIa),R⁵, R⁶ and R⁷ are H. In an exemplary embodiment, in the compound ofFormulae (II) or (IIa), R⁸, R⁹ and R¹⁰ are H. In an exemplaryembodiment, in the compound of Formulae (II) or (IIa), R¹¹, R¹² and R¹³are H. In an exemplary embodiment, in the compound of Formulae (II) or(IIa), R¹⁴, R¹⁵ and R¹⁶ are H.

In another exemplary embodiment, L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹ andL¹⁰ are members independently selected from substituted or unsubstitutedC₁ to C₆ alkylene. Exemplary compounds include those in which L¹, L²,L³, L⁴, L⁵, L⁶, L⁷, L, L⁹ and L¹⁰ are members independently selectedfrom substituted or unsubstituted ethylene. An exemplary ligandaccording to this embodiment has the structure of compound 3:

Functional Moiety

In one exemplary embodiment, the compounds of the invention (e.g. ligand3) are derivatized with a functional moiety. The functional moiety can,for example, be attached to one of the linker units or to one of thebuilding blocks. When two or more functional moieties are used, each canbe attached to any of the available linking sites.

In an exemplary embodiment, the functional moiety has the structure:

wherein L¹¹ is a linker moiety, which is a member selected fromsubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl and substituted or unsubstituted heteroaryl; and Xis a member selected from a reactive functional group and a targetingmoiety.

The functional moiety is preferably attached, so that the resultingfunctionalized ligand will be able to undergo formation of stable metalion complexes. In an exemplary embodiment, the macrocyclic ligand 3 isderivatized with a functional moiety. FIG. 2 shows preferredderivatization sites for 3.

FIG. 2

In one exemplary embodiment, compound 3 is derivatized at position (aa),(bb) or (cc) in FIG. 2. However, ligands, in which alternative positionswithin the core structure of the ligand (e.g., positions (dd) and (ee))are derivatized with a functional moiety, are expected to have similarlyuseful properties.

In an exemplary embodiment, the compound comprises one functionalmoiety. In another exemplary embodiment, the compound has a structurewhich is a member selected from:

In an exemplary embodiment, in the compound of Formulae (III) or (IIIa)or (IV) or (IVa) or (V) or (Va) or (VI) or (VIa) or (VII) or (VIIa) or(VIII) or (VIIIa), L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹ and L¹⁰ aremembers independently selected from substituted or unsubstituted C₁ toC₆ alkylene. In an exemplary embodiment, in the compound of Formulae(III) or (IIIa) or (IV) or (IVa) or (V) or (Va) or (VI) or (VIa) or(VII) or (VIIa) or (VIII) or (VIIIa), L¹, L², L⁴, L⁵ are membersindependently selected from substituted or unsubstituted ethylene. In anexemplary embodiment, in the compound of Formulae (III) or (IIIa) or(IV) or (IVa) or (V) or (Va) or (VI) or (VIa) or (VII) or (VIIa) or(VIII) or (VIIIa), L⁶, L⁷, L⁹, L¹⁰ are members independently selectedfrom substituted or unsubstituted ethylene. In an exemplary embodiment,in the compound of Formulae (III) or (IIIa) or (IV) or (IVa) or (V) or(Va) or (VI) or (VIa) or (VII) or (VIIa) or (VIII) or (VIIIa), L⁶, L⁷,L⁹, L¹⁰ are members independently selected from substituted orunsubstituted methylene. In an exemplary embodiment, in the compound ofFormulae (III) or (IIIa) or (IV) or (IVa) or (V) or (Va) or (VI) or(VIa) or (VII) or (VIIa) or (VIII) or (VIIIa), L³ is substituted orunsubstituted ethylene. In an exemplary embodiment, in the compound ofFormulae (II) or (IIa), L⁸ is substituted or unsubstituted ethylene. Inan exemplary embodiment, in the compound of Formulae (III) or (IIIa) or(IV) or (IVa) or (V) or (Va) or (VI) or (VIa) or (VII) or (VIIa) or(VIII) or (VIIIa), L¹, L², L³, L⁴ and L⁵ are substituted orunsubstituted ethylene. In an exemplary embodiment, in the compound ofFormulae (III) or (IIIa) or (IV) or (IVa) or (V) or (Va) or (VI) or(VIa) or (VII) or (VIa) or (VIII) or (VIIIa), L⁶, L⁷, L⁸, L⁹, L¹⁰ aremembers independently selected from substituted or unsubstitutedethylene. In an exemplary embodiment, in the compound of Formulae (III)or (IIIa) or (IV) or (IVa) or (V) or (Va) or (VI) or (VIa) or (VII) or(VIIa) or (VIII) or (VIIIa), L⁶, L⁷, L⁸, L⁹, L¹⁰ are membersindependently selected from substituted or unsubstituted methylene. Inan exemplary embodiment, in a compound described in this paragraph, thecomposition further comprises a metal, thus forming a complex. In anexemplary embodiment, in a compound described in this paragraph, themetal is a lanthanide. In an exemplary embodiment, in a compounddescribed in this paragraph, the lanthanide is a member selected fromNd, Sm, Eu, Tb, Dy and Yb. In an exemplary embodiment, in a compounddescribed in this paragraph, the lanthanide is Tb. In an exemplaryembodiment, in a compound described in this paragraph, the lanthanide isEu. In another exemplary embodiment, in a compound or complex describedin this paragraph, X is a targeting moiety which is a biomolecule. Inanother exemplary embodiment, in a compound or complex described in thisparagraph, X is a targeting moiety which is a biomolecule which is amember selected from a small-molecule ligand, a peptide, a protein, anenzyme, an antibody, an antigen, a nucleic acid, a carbohydrate, a lipidand a pharmacologically active molecule. In another exemplaryembodiment, in a compound or complex described in this paragraph, X is atargeting moiety which is a peptide, a protein, an enzyme or anantibody. In another exemplary embodiment, in a compound or complexdescribed in this paragraph, X is targeting moiety which is a nucleicacid. In another exemplary embodiment, in a compound or complexdescribed in this paragraph, X is a targeting moiety which is acarbohydrate. In another exemplary embodiment, in a terbium or europiumcomplex described in this paragraph, X is a targeting moiety which is abiomolecule which is a member selected from a small-molecule ligand, apeptide, a protein, an enzyme, an antibody, an antigen, a nucleic acid,a carbohydrate, a lipid and a pharmacologically active molecule. Inanother exemplary embodiment, in a terbium or europium complex describedin this paragraph, X is a targeting moiety which is a peptide, aprotein, an enzyme or an antibody. In another exemplary embodiment, in aterbium or europium complex described in this paragraph, X is atargeting moiety which is a nucleic acid. In another exemplaryembodiment, in a terbium or europium complex described in thisparagraph, X is a targeting moiety which is a carbohydrate.

In an exemplary embodiment, in the compound of Formulae (III) or (IIIa)or (IV) or (IVa) or (V) or (Va) or (VI) or (VIa) or (VII) or (VIIa) or(VIII) or (VIIIa), R⁵, R⁶ and R⁷ are H. In an exemplary embodiment, inthe compound of Formulae (III) or (IIIa) or (IV) or (IVa) or (V) or (Va)or (VI) or (VIa) or (VII) or (VIIa) or (VIII) or (VIIIa), R⁸, R⁹ and R¹⁰are H. In an exemplary embodiment, in the compound of Formulae (III) or(IIIa) or (IV) or (IVa) or (V) or (Va) or (VI) or (VIa) or (VII) or(VIa) or (VIII) or (VIIIa), R¹¹, R¹² and R¹³ are H. In an exemplaryembodiment, in the compound of Formulae (III) or (IIIa) or (IV) or (IVa)or (V) or (Va) or (VI) or (VIa) or (VII) or (VIIa) or (VIII) or (VIIIa),R¹⁴, R¹⁵ and R¹⁶ are H. In an exemplary embodiment, in a compounddescribed in this paragraph, the composition further comprises a metal,thus forming a complex. In an exemplary embodiment, in a compounddescribed in this paragraph, the metal is a lanthanide. In an exemplaryembodiment, in a compound described in this paragraph, the lanthanide isa member selected from Nd, Sm, Eu, Tb, Dy and Yb. In an exemplaryembodiment, in a compound described in this paragraph, the lanthanide isTb. In an exemplary embodiment, in a compound described in thisparagraph, the lanthanide is Eu. In another exemplary embodiment, in acompound or complex described in this paragraph, X is a targeting moietywhich is a biomolecule. In another exemplary embodiment, in a compoundor complex described in this paragraph, X is a targeting moiety which isa biomolecule which is a member selected from a small-molecule ligand, apeptide, a protein, an enzyme, an antibody, an antigen, a nucleic acid,a carbohydrate, a lipid and a pharmacologically active molecule. Inanother exemplary embodiment, in a compound or complex described in thisparagraph, X is a targeting moiety which is a peptide, a protein, anenzyme or an antibody. In another exemplary embodiment, in a compound orcomplex described in this paragraph, X is a targeting moiety which is anucleic acid. In another exemplary embodiment, in a compound or complexdescribed in this paragraph, X is a targeting moiety which is acarbohydrate. In another exemplary embodiment, in a terbium or europiumcomplex described in this paragraph, X is a targeting moiety which is abiomolecule which is a member selected from a small-molecule ligand, apeptide, a protein, an enzyme, an antibody, an antigen, a nucleic acid,a carbohydrate, a lipid and a pharmacologically active molecule. Inanother exemplary embodiment, in a terbium or europium complex describedin this paragraph, X is a targeting moiety which is a peptide, aprotein, an enzyme or an antibody. In another exemplary embodiment, in aterbium or europium complex described in this paragraph, X is atargeting moiety which is a nucleic acid. In another exemplaryembodiment, in a terbium or europium complex described in thisparagraph, X is a targeting moiety which is a carbohydrate.

In an exemplary embodiment, the compound comprises two functionalmoieties. In another exemplary embodiment, one of these functionalgroups is attached to L⁴ and the other functional group is attached to amember selected from L¹, L² and L⁵. In another exemplary embodiment, oneof these functional groups is attached to L⁴ and the other functionalgroup is attached to a member selected from L⁶, L⁷, L⁹ and L¹⁰. Inanother exemplary embodiment, one of these functional groups is attachedto L⁴ and the other functional group is attached to a member selectedfrom L³ and L⁸. In another exemplary embodiment, one of these functionalgroups is attached to L⁴ and the other functional group is attached to amember selected from R⁶, R⁹, R¹² and R¹⁵. In an exemplary embodiment, ina compound described in this paragraph, the composition furthercomprises a metal, thus forming a complex. In an exemplary embodiment,in a compound described in this paragraph, the metal is a lanthanide. Inan exemplary embodiment, in a compound described in this paragraph, thelanthanide is a member selected from Nd, Sm, Eu, Tb, Dy and Yb. In anexemplary embodiment, in a compound described in this paragraph, thelanthanide is Tb. In an exemplary embodiment, in a compound described inthis paragraph, the lanthanide is Eu. In another exemplary embodiment,in a compound or complex described in this paragraph, X is a targetingmoiety which is a biomolecule. In another exemplary embodiment, in acompound or complex described in this paragraph, X is a targeting moietywhich is a biomolecule which is a member selected from a small-moleculeligand, a peptide, a protein, an enzyme, an antibody, an antigen, anucleic acid, a carbohydrate, a lipid and a pharmacologically activemolecule. In another exemplary embodiment, in a compound or complexdescribed in this paragraph, X is a targeting moiety which is a peptide,a protein, an enzyme or an antibody. In another exemplary embodiment, ina compound or complex described in this paragraph, X is a targetingmoiety which is a nucleic acid. In another exemplary embodiment, in acompound or complex described in this paragraph, X is a targeting moietywhich is a carbohydrate. In another exemplary embodiment, in a terbiumor europium complex described in this paragraph, X is a targeting moietywhich is a biomolecule which is a member selected from a small-moleculeligand, a peptide, a protein, an enzyme, an antibody, an antigen, anucleic acid, a carbohydrate, a lipid and a pharmacologically activemolecule. In another exemplary embodiment, in a terbium or europiumcomplex described in this paragraph, X is a targeting moiety which is apeptide, a protein, an enzyme or an antibody. In another exemplaryembodiment, in a terbium or europium complex described in thisparagraph, X is a targeting moiety which is a nucleic acid. In anotherexemplary embodiment, in a terbium or europium complex described in thisparagraph, X is a targeting moiety which is a carbohydrate.

In another exemplary embodiment, one of these functional groups isattached to L³ and the other functional group is attached to a memberselected from L¹, L², L⁴ and L⁵. In another exemplary embodiment, one ofthese functional groups is attached to L³ and the other functional groupis attached to a member selected from L⁶, L⁷, L⁹ and L¹⁰. In anotherexemplary embodiment, one of these functional groups is attached to L³and the other functional group is attached to L⁸. In another exemplaryembodiment, one of these functional groups is attached to L³ and theother functional group is attached to a member selected from R⁶, R⁹, R¹²and R¹⁵. In an exemplary embodiment, in a compound described in thisparagraph, the composition further comprises a metal, thus forming acomplex. In an exemplary embodiment, in a compound described in thisparagraph, the metal is a lanthanide. In an exemplary embodiment, in acompound described in this paragraph, the lanthanide is a memberselected from Nd, Sm, Eu, Tb, Dy and Yb. In an exemplary embodiment, ina compound described in this paragraph, the lanthanide is Tb. In anexemplary embodiment, in a compound described in this paragraph, thelanthanide is Eu. In another exemplary embodiment, in a compound orcomplex described in this paragraph, X is a targeting moiety which is abiomolecule. In another exemplary embodiment, in a compound or complexdescribed in this paragraph, X is a targeting moiety which is abiomolecule which is a member selected from a small-molecule ligand, apeptide, a protein, an enzyme, an antibody, an antigen, a nucleic acid,a carbohydrate, a lipid and a pharmacologically active molecule. Inanother exemplary embodiment, in a compound or complex described in thisparagraph, X is a targeting moiety which is a peptide, a protein, anenzyme or an antibody. In another exemplary embodiment, in a compound orcomplex described in this paragraph, X is a targeting moiety which is anucleic acid. In another exemplary embodiment, in a compound or complexdescribed in this paragraph, X is a targeting moiety which is acarbohydrate. In another exemplary embodiment, in a terbium or europiumcomplex described in this paragraph, X is a targeting moiety which is abiomolecule which is a member selected from a small-molecule ligand, apeptide, a protein, an enzyme, an antibody, an antigen, a nucleic acid,a carbohydrate, a lipid and a pharmacologically active molecule. Inanother exemplary embodiment, in a terbium or europium complex describedin this paragraph, X is a targeting moiety which is a peptide, aprotein, an enzyme or an antibody. In another exemplary embodiment, in aterbium or europium complex described in this paragraph, X is atargeting moiety which is a nucleic acid. In another exemplaryembodiment, in a terbium or europium complex described in thisparagraph, X is a targeting moiety which is a carbohydrate.

In another exemplary embodiment, the compound of Formulae (III) or(IIIa) or (IV) or (IVa) or (V) or (Va) or (VI) or (VIa) or (VII) or(VIIa) or (VIII) or (VIIIa), in which X is a member selected from anamine, a carboxylic acid, a maleimidyl, a thiazolidyl, a substituted orunsubstituted NHS ester, a sulfonated NHS ester and a succinimidylmoiety. In another exemplary embodiment, the compound of Formulae (III)or (IIIa) or (IV) or (IVa) or (V) or (Va) or (VI) or (VIa) or (VII) or(VIIa) or (VIII) or (VIIIa), in which L¹¹ is substituted orunsubstituted heteroalkylene or substituted or unsubstituted alkyleneand X is a member selected from an amine, a carboxylic acid, amaleimidyl, a thiazolidyl, a substituted or unsubstituted NHS ester, asulfonated NHS ester and a succinimidyl moiety. In another exemplaryembodiment, the compound of Formulae (III) or (IIIa) or (IV) or (IVa) or(V) or (Va) or (VI) or (VIa) or (VII) or (VIIa) or (VIII) or (VIIIa),L¹¹-X is a member selected

In another exemplary embodiment, in the compound of Formulae (III) or(IIIa) or (IV) or (IVa) or (V) or (Va) or (VI) or (VIa) or (VII) or(VIIa) or (VIII) or (VIIIa), L¹¹ is substituted or unsubstitutedarylalkyl. In another exemplary embodiment, in the compound of Formulae(III) or (IIIa) or (IV) or (IVa) or (V) or (Va) or (VI) or (VIa) or(VII) or (VIIa) or (VIII) or (VIIIa), L¹¹ is substituted orunsubstituted arylalkyl and X is a member selected from an amine, acarboxylic acid, a maleimidyl, a thiazolidyl, a substituted orunsubstituted NHS ester, a sulfonated NHS ester and a succinimidylmoiety. In an exemplary embodiment, in a compound described in thisparagraph, the composition further comprises a metal, thus forming acomplex. In an exemplary embodiment, in a compound described in thisparagraph, the metal is a lanthanide. In an exemplary embodiment, in acompound described in this paragraph, the lanthanide is Tb. In anexemplary embodiment, in a compound described in this paragraph, thelanthanide is Eu.

In another exemplary embodiment, the compound of Formulae (III) or (IIa)or (IV) or (IVa) or (V) or (Va) or (VI) or (VIa) or (VII) or (VIIa) or(VIII) or (VIIIa), in which L¹¹ is substituted or unsubstitutedheteroalkylene or substituted or unsubstituted alkylene and X is atargeting moiety which is a biomolecule. In another exemplaryembodiment, the compound of Formulae (III) or (IIIa) or (IV) or (IVa) or(V) or (Va) or (VI) or (VIa) or (VII) or (VIIa) or (VIII) or (VIIIa), inwhich L¹¹ is substituted or unsubstituted C₁-C₆ alkylene and X is atargeting moiety which is a biomolecule. In another exemplaryembodiment, the compound of Formulae (III) or (IIIa) or (IV) or (IVa) or(V) or (Va) or (VI) or (VIa) or (VII) or (VIIa) or (VIII) or (VIIIa), inwhich L¹¹ is substituted or unsubstituted butylene and X is a targetingmoiety which is a biomolecule. In another exemplary embodiment, thecompound of Formulae (III) or (IIIa) or (IV) or (IVa) or (V) or (Va) or(VI) or (VIa) or (VII) or (VIa) or (VIII) or (VIIIa), in which L¹¹ isunsubstituted butylene and X is a targeting moiety which is abiomolecule. In another exemplary embodiment, in a compound described inthis paragraph, the composition further comprises a metal, thus forminga complex. In an exemplary embodiment, in a compound described in thisparagraph, the metal is a lanthanide. In an exemplary embodiment, in acompound described in this paragraph, the lanthanide is Tb. In anexemplary embodiment, in a compound described in this paragraph, thelanthanide is Eu. In another exemplary embodiment, in a compound orcomplex described in this paragraph, X is a targeting moiety which is abiomolecule which is a member selected from a small-molecule ligand, apeptide, a protein, an enzyme, an antibody, an antigen, a nucleic acid,a carbohydrate, a lipid and a pharmacologically active molecule. Inanother exemplary embodiment, in a compound or complex described in thisparagraph, X is a targeting moiety which is a peptide, a protein, anenzyme or an antibody. In another exemplary embodiment, in a compound orcomplex described in this paragraph, X is a targeting moiety which is anucleic acid. In another exemplary embodiment, in a compound or complexdescribed in this paragraph, X is a targeting moiety which is acarbohydrate. In another exemplary embodiment, in a terbium or europiumcomplex described in this paragraph, X is a targeting moiety which is abiomolecule which is a member selected from a small-molecule ligand, apeptide, a protein, an enzyme, an antibody, an antigen, a nucleic acid,a carbohydrate, a lipid and a pharmacologically active molecule. Inanother exemplary embodiment, in a terbium or europium complex describedin this paragraph, X is a targeting moiety which is a peptide, aprotein, an enzyme or an antibody. In another exemplary embodiment, in aterbium or europium complex described in this paragraph, X is atargeting moiety which is a nucleic acid. In another exemplaryembodiment, in a terbium or europium complex described in thisparagraph, X is a targeting moiety which is a carbohydrate.

In an exemplary embodiment, in the compound of Formulae (III) or (IIIa)or (IV) or (IVa) or (V) or (Va) or (VI) or (VIa) or (VII) or (VIIa) or(VIII) or (VIIIa), L¹, L², L⁴ and L⁵ are members independently selectedfrom substituted or unsubstituted ethylene, and L¹¹ is substituted orunsubstituted heteroalkylene or substituted or unsubstituted alkyleneand X is a targeting moiety which is a biomolecule. In another exemplaryembodiment, in a compound described in this paragraph, the compositionfurther comprises a metal, thus forming a complex. In an exemplaryembodiment, in a compound described in this paragraph, the metal is alanthanide. In an exemplary embodiment, in a compound described in thisparagraph, the lanthanide is Tb. In an exemplary embodiment, in acompound described in this paragraph, the lanthanide is Eu. In anotherexemplary embodiment, in a compound or complex described in thisparagraph, X is a targeting moiety which is a biomolecule which is amember selected from a small-molecule ligand, a peptide, a protein, anenzyme, an antibody, an antigen, a nucleic acid, a carbohydrate, a lipidand a pharmacologically active molecule. In another exemplaryembodiment, in a compound or complex described in this paragraph, X is atargeting moiety which is a peptide, a protein, an enzyme or anantibody. In another exemplary embodiment, in a compound or complexdescribed in this paragraph, X is a targeting moiety which is a nucleicacid. In another exemplary embodiment, in a compound or complexdescribed in this paragraph, X is a targeting moiety which is acarbohydrate. In another exemplary embodiment, in a terbium or europiumcomplex described in this paragraph, X is a targeting moiety which is abiomolecule which is a member selected from a small-molecule ligand, apeptide, a protein, an enzyme, an antibody, an antigen, a nucleic acid,a carbohydrate, a lipid and a pharmacologically active molecule. Inanother exemplary embodiment, in a terbium or europium complex describedin this paragraph, X is a targeting moiety which is a peptide, aprotein, an enzyme or an antibody. In another exemplary embodiment, in aterbium or europium complex described in this paragraph, X is atargeting moiety which is a nucleic acid. In another exemplaryembodiment, in a terbium or europium complex described in thisparagraph, X is a targeting moiety which is a carbohydrate.

Preferred compounds of the invention that include a functional moietyhave the structure:

wherein L¹¹, X, R⁵, R⁶, R⁷, R⁸, R⁹ R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶are as defined above. In an exemplary embodiment, L¹¹ is a memberselected from substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl and substituted or unsubstituted aryl and X isa targeting moiety which is a biomolecule. In an exemplary embodiment,L¹¹ is a member selected from substituted or unsubstitutedheteroalkylene or substituted or unsubstituted alkylene and X is atargeting moiety which is a biomolecule which is a member selected froma small-molecule ligand, a peptide, a protein, an enzyme, an antibody,an antigen, a nucleic acid, a carbohydrate, a lipid and apharmacologically active molecule. In an exemplary embodiment, L¹¹ issubstituted or unsubstituted butylene and X is an antibody, an enzyme, anucleic acid, a carbohydrate. In another exemplary embodiment, in acompound described in this paragraph, the composition further comprisesa metal, thus forming a complex. In an exemplary embodiment, in acompound described in this paragraph, the metal is a lanthanide. In anexemplary embodiment, in a compound described in this paragraph, thelanthanide is Tb. In an exemplary embodiment, in a compound described inthis paragraph, the lanthanide is Eu.

For instance, functionalization of compound 3 at position (aa) (FIG. 2)with a (CH₂)₄NH₂ group leads to the macrocyclic derivative 4:

Reactive Functional Groups

In one embodiment, the functional moiety includes a reactive functionalgroup, X, which can be used to covalently attach the complexing agent toanother molecule. In another exemplary embodiment, the other molecule isa biomolecule. In another exemplary embodiment, the other molecule asmall-molecule ligand, a peptide, a protein, an enzyme, an antibody, anantigen, a nucleic acid, a carbohydrate, a lipid and a pharmacologicallyactive molecule. Alternatively, the reactive functional group can beused to link the ligand to a nano-particle of any kind.

Reactive functional groups and classes of reactions useful in attachingthe compounds described herein are generally those that are well knownin the art of bioconjugate chemistry. Currently favored classes ofreactions available with reactive functional groups of the invention arethose which proceed under relatively mild conditions. These include, butare not limited to nucleophilic substitutions (e.g., reactions of aminesand alcohols with acyl halides and activated esters), electrophilicsubstitutions (e.g., enamine reactions) and additions to carbon-carbonand carbon-heteroatom multiple bonds (e.g., Michael reactions andDiels-Alder reactions). These and other useful reactions are discussed,for example, in: March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley& Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, AcademicPress, San Diego, 1996; and Feeney et al., MODIFICATION OF PROTEINS;Advances in Chemistry Series, Vol. 198, American Chemical Society,Washington, D.C., 1982.

a) Amines and Amino-Reactive Groups

In one embodiment, the reactive functional group is a member selectedfrom amines, such as a primary or secondary amine, hydrazines,hydrazides, and sulfonylhydrazides. Amines can, for example, beacylated, alkylated or oxidized. Useful non-limiting examples ofamino-reactive groups include N-hydroxysuccinimide (NHS) esters,sulfo-NHS esters, imidoesters, isocyanates, isothiocyanates,acylhalides, arylazides, p-nitrophenyl esters, aldehydes, sulfonylchlorides and carboxyl groups.

NHS esters and sulfo-NHS esters react preferentially with the primary(including aromatic) amino groups of the reaction partner. The imidazolegroups of histidines are known to compete with primary amines forreaction, but the reaction products are unstable and readily hydrolyzed.The reaction involves the nucleophilic attack of an amine on the acidcarboxyl of an NHS ester to form an amide, releasing theN-hydroxysuccinimide.

Imidoesters are the most specific acylating reagents for reaction withthe amine groups of e.g., a protein. At a pH between 7 and 10,imidoesters react only with primary amines. Primary amines attackimidates nucleophilically to produce an intermediate that breaks down toamidine at high pH or to a new imidate at low pH. The new imidate canreact with another primary amine, thus crosslinking two amino groups, acase of a putatively monofunctional imidate reacting bifunctionally. Theprincipal product of reaction with primary amines is an amidine that isa stronger base than the original amine. The positive charge of theoriginal amino group is therefore retained. As a result, imidoesters donot affect the overall charge of the conjugate.

Isocyanates (and isothiocyanates) react with the primary amines of theconjugate components to form stable bonds. Their reactions withsulfhydryl, imidazole, and tyrosyl groups give relatively unstableproducts.

Acylazides are also used as amino-specific reagents in whichnucleophilic amines of the reaction partner attack acidic carboxylgroups under slightly alkaline conditions, e.g. pH 8.5.

Arylhalides such as 1,5-difluoro-2,4-dinitrobenzene react preferentiallywith the amino groups and tyrosine phenolic groups of the conjugatecomponents, but also with its sulfhydryl and imidazole groups.

p-Nitrophenyl esters of carboxylic acids are also useful amino-reactivegroups. Although the reagent specificity is not very high, α- andε-amino groups appear to react most rapidly.

Aldehydes react with primary amines of the conjugate components (e.g.,ε-amino group of lysine residues). Although unstable, Schiff bases areformed upon reaction of the protein amino groups with the aldehyde.Schiff bases, however, are stable, when conjugated to another doublebond. The resonant interaction of both double bonds prevents hydrolysisof the Schiff linkage. Furthermore, amines at high local concentrationscan attack the ethylenic double bond to form a stable Michael additionproduct. Alternatively, a stable bond may be formed by reductiveamination.

Aromatic sulfonyl chlorides react with a variety of sites of theconjugate components, but reaction with the amino groups is the mostimportant, resulting in a stable sulfonamide linkage.

Free carboxyl groups react with carbodiimides, soluble in both water andorganic solvents, forming pseudoureas that can then couple to availableamines yielding an amide linkage. Yamada et al., Biochemistry 1981, 20:4836-4842, e.g., teach how to modify a protein with carbodiimides.

b) Sulfhydryl and Sulfhydryl-Reactive Groups

In another embodiment, the reactive functional group is a memberselected from a sulfhydryl group (which can be converted to disulfides)and sulfhydryl-reactive groups. Useful non-limiting examples ofsulfhydryl-reactive groups include maleimides, alkyl halides, acylhalides (including bromoacetamide or chloroacetamide), pyridyldisulfides, and thiophthalimides.

Maleimides react preferentially with the sulfhydryl group of theconjugate components to form stable thioether bonds. They also react ata much slower rate with primary amino groups and the imidazole groups ofhistidines. However, at pH 7 the maleimide group can be considered asulfhydryl-specific group, since at this pH the reaction rate of simplethiols is 1000-fold greater than that of the corresponding amine.

Alkyl halides react with sulfhydryl groups, sulfides, imidazoles, andamino groups. At neutral to slightly alkaline pH, however, alkyl halidesreact primarily with sulfhydryl groups to form stable thioether bonds.At higher pH, reaction with amino groups is favored.

Pyridyl disulfides react with free sulfhydryl groups via disulfideexchange to give mixed disulfides. As a result, pyridyl disulfides arerelatively specific sulfhydryl-reactive groups.

Thiophthalimides react with free sulfhydryl groups to also formdisulfides.

c) Other Reactive Functional Groups

Other exemplary reactive functional groups include:

-   -   (a) carboxyl groups and various derivatives thereof including,        but not limited to, N-hydroxybenztriazole esters, acid halides,        acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl,        alkenyl, alkynyl and aromatic esters;    -   (b) hydroxyl groups, which can be converted to esters, ethers,        aldehydes, etc.;    -   (c) haloalkyl groups, wherein the halide can be displaced with a        nucleophilic group such as, for example, an amine, a carboxylate        anion, thiol anion, carbanion, or an alkoxide ion, thereby        resulting in the covalent attachment of a new group at the site        of the halogen atom;    -   (d) dienophile groups, which are capable of participating in        Diels-Alder reactions such as, for example, maleimido groups;    -   (e) aldehyde or ketone groups, such that subsequent        derivatization is possible via formation of carbonyl derivatives        such as, for example, imines, hydrazones, semicarbazones or        oximes, or via such mechanisms as Grignard addition or        alkyllithium addition;    -   (f) alkenes, which can undergo, for example, cycloadditions,        acylation, Michael addition, etc;    -   (g) epoxides, which can react with, for example, amines and        hydroxyl groups;    -   (h) phosphoramidites and other standard functional groups useful        in nucleic acid synthesis and    -   (i) any other functional group useful to form a covalent bond        between the functionalized ligand and a molecular entity or a        surface.        d) Reactive Functional Groups with Non-Specific Reactivities

In addition to the use of site-specific reactive moieties, the presentinvention contemplates the use of non-specific reactive functionalgroups to link a compound described herein to a targeting moiety.Non-specific groups include photoactivatable groups, for example.Photoactivatable groups are ideally inert in the dark and are convertedto reactive species in the presence of light. In one embodiment,photoactivatable groups are selected from precursors of nitrenesgenerated upon heating or photolysis of azides. Electron-deficientnitrenes are extremely reactive and can react with a variety of chemicalbonds including N—H, O—H, C—H, and C═C. Although three types of azides(aryl, alkyl, and acyl derivatives) may be employed, arylazides arepresently preferred. The reactivity of arylazides upon photolysis isbetter with N—H and O—H than C—H bonds. Electron-deficient arylnitrenesrapidly ring-expand to form dehydroazepines, which tend to react withnucleophiles, rather than form C—H insertion products. The reactivity ofarylazides can be increased by the presence of electron-withdrawingsubstituents such as nitro or hydroxyl groups in the ring. Suchsubstituents push the absorption maximum of arylazides to longerwavelength. Unsubstituted arylazides have an absorption maximum in therange of 260-280 nm, while hydroxy and nitroarylazides absorbsignificant light beyond 305 nm. Therefore, hydroxy and nitroarylazidesare most preferable since they allow to employ less harmful photolysisconditions for the affinity component than unsubstituted arylazides.

In another preferred embodiment, photoactivatable groups are selectedfrom fluorinated arylazides. The photolysis products of fluorinatedarylazides are arylnitrenes, all of which undergo the characteristicreactions of this group, including C—H bond insertion, with highefficiency (Keana et al, J. Org. Chem. 55: 3640-3647, 1990).

In another embodiment, photoactivatable groups are selected frombenzophenone residues. Benzophenone reagents generally give highercrosslinking yields than arylazide reagents.

In another embodiment, photoactivatable groups are selected from diazocompounds, which form an electron-deficient carbene upon photolysis.These carbenes undergo a variety of reactions including insertion intoC—H bonds, addition to double bonds (including aromatic systems),hydrogen attraction and coordination to nucleophilic centers to givecarbon ions.

In still another embodiment, photoactivatable groups are selected fromdiazopyruvates. For example, the p-nitrophenyl ester of p-nitrophenyldiazopyruvate reacts with aliphatic amines to give diazopyruvic acidamides that undergo ultraviolet photolysis to form aldehydes. Thephotolyzed diazopyruvate-modified affinity component will react likeformaldehyde or glutaraldehyde forming intraprotein crosslinks.

It is well within the abilities of a person skilled in the art to selecta reactive functional group, according to the reaction partner. As anexample, an activated ester, such as an NHS ester will be useful tolabel a protein via lysine residues. Sulfhydryl reactive groups, such asmaleimides can be used to label proteins via amino acid residuescarrying an SH-group (e.g., cysteine). Antibodies may be labeled byfirst oxidizing their carbohydrate moieties (e.g., with periodate) andreacting resulting aldehyde groups with a hydrazine containing ligand.

Additional exemplary combinations of reactive functional groups found ona compound of the invention and on a targeting moiety (or polymer orlinker) are set forth in Table 2.

TABLE 2 Chemical Chemical Functionality 1 Functionality 2 LinkageHydroxy Carboxy Ester Hydroxy Carbonate Amine Carbamate SO₃ Sulfate PO₃Phosphate Carboxy Acyloxyalkyl Ketone Ketal Aldehyde Acetal HydroxyAnhydride Mercapto Mercapto Disulfide Carboxy Acyloxyalkyl ThioetherCarboxy Thioester Carboxy Amino amide Mercapto Thioester CarboxyAcyloxyalkyl ester Carboxy Acyloxyalkyl amide Amino Acyloxyalkoxycarbonyl Carboxy Anhydride Carboxy N-acylamide Hydroxy Ester HydroxyHydroxymethyl ketone ester Hydroxy Alkoxycarbonyl oxyalkyl Amino CarboxyAcyloxyalkylamine Carboxy Acyloxyalkylamide Amino Urea Carboxy AmideCarboxy Acyloxyalkoxycarbonyl Amide N-Mannich base Carboxy Acyloxyalkylcarbamate Phosphate Hydroxy Phosphate oxygen ester Amine PhosphoramidateMercapto Thiophosphate ester Ketone Carboxy Enol ester SulfonamideCarboxy Acyloxyalkyl sulfonamide Ester N-sulfonyl-imidate

One skilled in the art will readily appreciate that many of theselinkages may be produced in a variety of ways and using a variety ofconditions. For the preparation of esters, see, e.g., March supra at1157; for thioesters, see, March, supra at 362-363, 491, 720-722, 829,941, and 1172; for carbonates, see, March, supra at 346-347; forcarbamates, see, March, supra at 1156-57; for amides, see, March supraat 1152; for ureas and thioureas, see, March supra at 1174; for acetalsand ketals, see, Greene et al. supra 178-210 and March supra at 1146;for acyloxyalkyl derivatives, see, PRODRUGS: TOPICAL AND OCULAR DRUGDELIVERY, K. B. Sloan, ed., Marcel Dekker, Inc., New York, 1992; forenol esters, see, March supra at 1160; for N-sulfonylimidates, see,Bundgaard et al., J. Med. Chem., 31:2066 (1988); for anhydrides, see,March supra at 355-56, 636-37, 990-91, and 1154; for N-acylamides, see,March supra at 379; for N-Mannich bases, see, March supra at 800-02, and828; for hydroxymethyl ketone esters, see, Petracek et al. Annals NYAcad. Sci., 507:353-54 (1987); for disulfides, see, March supra at 1160;and for phosphonate esters and phosphonamidates.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the reactions necessary to assemblethe reactive ligand analogue. Alternatively, a reactive functional groupcan be protected from participating in the reaction by the presence of aprotecting group. Those of skill in the art will understand how toprotect a particular functional group from interfering with a chosen setof reaction conditions. For examples of useful protecting groups, seeGreene et al., PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, John Wiley &Sons, New York, 1991.

Generally, prior to forming the linkage between the compound of theinvention and the targeting (or other) agent, and optionally, the linkergroup, at least one of the chemical functionalities will be activated.One skilled in the art will appreciate that a variety of chemicalfunctionalities, including hydroxy, amino, and carboxy groups, can beactivated using a variety of standard methods and conditions. Forexample, a hydroxyl group of the ligand (or targeting agent) can beactivated through treatment with phosgene to form the correspondingchloroformate, or p-nitrophenylchloroformate to form the correspondingcarbonate.

In an exemplary embodiment, the invention makes use of a targeting agentthat includes a carboxyl functionality. Carboxyl groups may be activatedby, for example, conversion to the corresponding acyl halide or activeester. This reaction may be performed under a variety of conditions asillustrated in March, supra pp. 388-89. In an exemplary embodiment, theacyl halide is prepared through the reaction of the carboxyl-containinggroup with oxalyl chloride. The activated agent is combined with aligand or ligand-linker arm combination to form a conjugate of theinvention. Those of skill in the art will appreciate that the use ofcarboxyl-containing targeting agents is merely illustrative, and thatagents having many other functional groups can be conjugated to theligands of the invention.

Targeting Moieties

In an exemplary embodiment, the targeting moiety is a biomolecule.Exemplary targeting moieties include small-molecule ligands, lipids,linear and cyclic peptides, polypeptides (e.g., EPO, insulin etc.) andproteins, such as enzymes and receptors. Other targeting moietiesinclude antibodies and antibody fragments (e.g., those generated torecognize small-molecules and receptor ligands), antigens, nucleic acids(e.g. RNA and cDNA), carbohydrate moieties (e.g., polysaccharides), andpharmacologically active molecules, such as toxins, pharmaceutical drugsand drugs of abuse (e.g. steroids). Additional targeting moieties areselected from solid supports and polymeric surfaces (e.g., polymericbeads and plastic sample reservoirs, such as plastic well-plates),sheets, fibers and membranes. Targeting moieties also include particles(e.g., nano-particles) and drug-delivery vehicles.

In one embodiment, the targeting moiety includes at least one unit of amacrocyclic compound. In an exemplary embodiment, the macrocycliccompound of the targeting moiety has a structure which is a compounddescribed herein. In another exemplary embodiment, the macrocycliccompound of the targeting moiety has a structure which according toFormula (I) or (Ia) or (II) or (IIa) or (III) or (IIIa) or (IV) or (IVa)or (V) or (Va) or (VI) or (VIa) or (VII) or (VIIa) or (VIII) or (VIIIa).In another exemplary embodiment, the compound of the invention has adendrimeric structure and encompasses several ligands having a structureaccording to a compound described herein. In another exemplaryembodiment, the compound of the invention has a dendrimeric structureand encompasses several ligands having a structure according to Formula(I) or (Ia) or (II) or (IIa) or (III) or (IIIa) or (IV) or (IVa) or (V)or (Va) or (VI) or (VIa) or (VII) or (VIIa) or (VIII) or (VIIIa). In afurther exemplary embodiment, according to this aspect, a complex basedon such dendrimer includes at least two metal ions.

In one exemplary embodiment, the targeting moiety is substituted with aluminescence modifying group that allows luminescence energy transferbetween a complex of the invention and the luminescence modifying groupwhen the complex is excited.

In further embodiments, the compounds of the invention can be used inany assay format aimed at detecting a lipid in a sample (e.g., in theblood of a patient). An exemplary complex according to this embodiment,includes a targeting moiety, which is a protein containing a lipidrecognition motif. Exemplary lipid binding proteins include those thatbind to phosphatidylinositol, phosphatidylinositol phosphates or otherbiological lipids.

In another example, the targeting moiety is an antibody that recognizesand binds to an analyte. In an exemplary assay system an analyte may bedetected in a sample by first incubating the sample with a complex ofthe invention, wherein the complex is covalently bound to an antibodythat includes a binding site for the analyte. To the mixture can then beadded an excess of a probe molecules that binds to the same binding siteas the analyte and includes a luminescence modifying group (e.g. anacceptor). The presence and concentration of analyte in the sample isindicated by the luminescence of the assay mixture. For instance, if theconcentration of analyte in the sample is high, many of the antibodybinding sites will be occupied with the analyte and less binding siteswill be available for the probe molecule. In an exemplary embodiment,the analyte is a lipid molecule.

In another preferred embodiment, the targeting moiety is a drug moiety.The drug moieties can be agents already accepted for clinical use orthey can be drugs whose use is experimental, or whose activity ormechanism of action is under investigation. In another preferredembodiment, the targeting moiety is a drug of abuse. The drug moietiescan have a proven action in a given disease state or can be onlyhypothesized to show desirable action in a given disease state. In apreferred embodiment, the drug moieties are compounds which are beingscreened for their ability to interact with an analyte of choice. Assuch, drug moieties which are useful as targeting moieties in theinstant invention include drugs from a broad range of drug classeshaving a variety of pharmacological activities.

Classes of useful agents include, for example, non-steroidalanti-inflammatory drugs (NSAIDS). The NSAIDS can, for example, beselected from the following categories: (e.g., propionic acidderivatives, acetic acid derivatives, fenamic acid derivatives,biphenylcarboxylic acid derivatives and oxicams); steroidalanti-inflammatory drugs including hydrocortisone and the like;antihistaminic drugs (e.g., chlorpheniramine, triprolidine); antitussivedrugs (e.g., dextromethorphan, codeine, carmiphen and carbetapentane);antipruritic drugs (e.g., methidilizine and trimeprizine);anticholinergic drugs (e.g., scopolamine, atropine, homatropine,levodopa); anti-emetic and antinauseant drugs (e.g., cyclizine,meclizine, chlorpromazine, buclizine); anorexic drugs (e.g.,benzphetamine, phentermine, chlorphentermine, fenfluramine); centralstimulant drugs (e.g., amphetamine, methamphetamine, dextroamphetamineand methylphenidate); antiarrhythmic drugs (e.g., propanolol,procainamide, disopyraminde, quinidine, encamide); .beta.-adrenergicblocker drugs (e.g., metoprolol, acebutolol, betaxolol, labetalol andtimolol); cardiotonic drugs (e.g., milrinone, aminone and dobutamine);antihypertensive drugs (e.g., enalapril, clonidine, hydralazine,minoxidil, guanadrel, guanethidine); diuretic drugs (e.g., amiloride andhydrochlorothiazide); vasodilator drugs (e.g., diltazem, amiodarone,isosuprine, nylidrin, tolazoline and verapamil); vasoconstrictor drugs(e.g., dihydroergotamine, ergotamine and methylsergide); antiulcer drugs(e.g., ranitidine and cimetidine); anesthetic drugs (e.g., lidocaine,bupivacaine, chlorprocaine, dibucaine); antidepressant drugs (e.g.,imipramine, desipramine, amitryptiline, nortryptiline); tranquilizer andsedative drugs (e.g., chlordiazepoxide, benacytyzine, benzquinamide,flurazapam, hydroxyzine, loxapine and promazine); antipsychotic drugs(e.g., chlorprothixene, fluphenazine, haloperidol, molindone,thioridazine and trifluoperazine); antimicrobial drugs (antibacterial,antifungal, antiprotozoal and antiviral drugs).

Antimicrobial drugs which are preferred for incorporation into thepresent composition include, for example, pharmaceutically acceptablesalts of .beta.-lactam drugs, quinolone drugs, ciprofloxacin,norfloxacin, tetracycline, erythromycin, amikacin, triclosan,doxycycline, capreomycin, chlorhexidine, chlortetracycline,oxytetracycline, clindamycin, ethambutol, hexamidine isothionate,metronidazole, pentamidine, gentamycin, kanamycin, lineomycin,methacycline, methenamine, minocycline, neomycin, netilmycin,paromomycin, streptomycin, tobramycin, miconazole and amanfadine.

Other drug moieties of use in practicing the present invention includeantineoplastic drugs (e.g., antiandrogens (e.g., leuprolide orflutamide), cytocidal agents (e.g., adriamycin, doxorubicin, taxol,cyclophosphamide, busulfan, cisplatin, .alpha.-2-interferon)anti-estrogens (e.g., tamoxifen), antimetabolites (e.g., fluorouracil,methotrexate, mercaptopurine, thioguanine).

The targeting moiety can also comprise hormones (e.g.,medroxyprogesterone, estradiol, leuprolide, megestrol, octreotide orsomatostatin); muscle relaxant drugs (e.g., cinnamedrine,cyclobenzaprine, flavoxate, orphenadrine, papaverine, mebeverine,idaverine, ritodrine, dephenoxylate, dantrolene and azumolen);antispasmodic drugs; bone-active drugs (e.g., diphosphonate andphosphonoalkylphosphinate drug compounds); endocrine modulating drugs(e.g., contraceptives (e.g., ethinodiol, ethinyl estradiol,norethindrone, mestranol, desogestrel, medroxyprogesterone), modulatorsof diabetes (e.g., glyburide or chlorpropamide), anabolics, such astestolactone or stanozolol, androgens (e.g., methyltestosterone,testosterone or fluoxymesterone), antidiuretics (e.g., desmopressin) andcalcitonins).

Also of use in the present invention are estrogens (e.g.,diethylstilbesterol), glucocorticoids (e.g., triamcinolone,betamethasone, etc.) and progenstogens, such as norethindrone,ethynodiol, norethindrone, levonorgestrel; thyroid agents (e.g.,liothyronine or levothyroxine) or anti-thyroid agents (e.g.,methimazole); antihyperprolactinemic drugs (e.g., cabergoline); hormonesuppressors (e.g., danazol or goserelin), oxytocics (e.g.,methylergonovine or oxytocin) and prostaglandins, such as mioprostol,alprostadil or dinoprostone, can also be employed.

Other useful targeting moieties include immunomodulating drugs (e.g.,antihistamines, mast cell stabilizers, such as lodoxamide and/orcromolyn, steroids (e.g., triamcinolone, beclomethazone, cortisone,dexamethasone, prednisolone, methylprednisolone, beclomethasone, orclobetasol), histamine H₂ antagonists (e.g., famotidine, cimetidine,ranitidine), immunosuppressants (e.g., azathioprine, cyclosporin), etc.Groups with anti-inflammatory activity, such as sulindac, etodolac,ketoprofen and ketorolac, are also of use. Other drugs of use inconjunction with the present invention will be apparent to those ofskill in the art.

The above enumerated, and other molecules, can be attached to thecompounds of the invention, to solid substrates and the like by methodswell-known to those of skill in the art. Ample guidance can be found inliterature devoted to, for example, the fields of bioconjugate chemistryand drug delivery. For example, one of skill, faced with a drugcomprising an available amine will be able to choose from among avariety of amine derivatizing reactions, locate an appropriatelyfunctionalized partner (e.g., a carboxylic acid terminated thiol) forthe organic layer and react the partners under conditions chosen toeffect the desired coupling (e.g., dehydrating agents, e.g.,dicyclohexylcarbodiimide). See, for example, MODIFICATION OF PROTEINS:FOOD, NUTRITIONAL, AND PHARMACOLOGICAL ASPECTS, Feeney et al., Eds.,American Chemical Society, Washington, D.C., 1982, pp. 370-387;POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, Dunn et al., Eds., AmericanChemical Society, Washington, D.C., 1991.

Linker L¹¹

In one preferred embodiment, the linker L¹¹ of the functional moiety islong enough to avoid side reactions during synthesis (e.g.intra-molecular reactions, such as intra-molecular peptide bondformation), to allow coupling of the compound or complex of theinvention to a targeting moiety and to allow the targeting moiety tofulfill its intended function. Useful linkers include those with about 2to about 50 linear atoms, preferably about 4 to about 20 linear atoms.

In another exemplary embodiment, the linker moiety L¹¹ or the targetingmoiety include a polyether, such as polyethylene glycol (PEG) andderivatives thereof. In one example, the polyether has a molecularweight between about 50 to about 10,000 daltons.

Exemplary Compounds

Exemplary compounds of the invention include:

wherein R¹, R², R³ and R⁴ are as defined above. In an exemplaryembodiment, one of these exemplary compounds is chelated to a metal,thus forming a complex. In an exemplary embodiment, in a compounddescribed in this paragraph, the metal is a lanthanide. In an exemplaryembodiment, the lanthanide is a member selected from Nd, Sm, Eu, Tb, Dyand Yb. In an exemplary embodiment, the lanthanide is Tb. In anexemplary embodiment, in a compound described in this paragraph, thelanthanide is Eu. In an exemplary embodiment, the compound is 4 and thelanthanide is a member selected from Nd, Sm, Eu, Tb, Dy and Yb. In anexemplary embodiment, the compound is 4 and the lanthanide is Tb. In anexemplary embodiment, the compound is 4 and the lanthanide is Eu. In anexemplary embodiment, the compound is 5a and the lanthanide is a memberselected from Nd, Sm, Eu, Tb, Dy and Yb. In an exemplary embodiment, thecompound is 5a and the lanthanide is Tb. In an exemplary embodiment, thecompound is 5a and the lanthanide is Eu.Synthesis

The compounds and complexes of the invention are synthesized by anappropriate combination of generally well-known synthetic methods.Techniques useful in synthesizing the compounds of the invention areboth readily apparent and accessible to those of skill in the relevantart. The discussion below is offered to illustrate certain of thediverse methods available for use in assembling the compounds of theinvention, it is not intended to limit the scope of reactions orreaction sequences that are useful in preparing the compounds of thepresent invention.

The compounds of the invention can be prepared as a single stereoisomeror as a mixture of stereoisomers. In a preferred embodiment, thecompounds are prepared as substantially a single isomer. Isomericallypure compounds are prepared by using synthetic intermediates that areisomerically pure in combination with reactions that either leave thestereochemistry at a chiral center unchanged or result in its completeinversion. Alternatively, the final product or intermediates along thesynthetic route can be resolved into a single stereoisomer. Techniquesfor inverting or leaving unchanged a particular stereocenter, and thosefor resolving mixtures of stereoisomers are well known in the art and itis well within the ability of one of skill in the art to choose anappropriate method for a particular situation. See, generally, Furnisset al. (eds.) VOGEL'S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY 5_(TH)ED., Longman Scientific and Technical Ltd., Essex, 1991, pp. 809-816;and Heller, Acc. Chem. Res. 23: 128 (1990).

In one embodiment, the compounds of the invention are synthesized byreacting a cap molecule with appropriate building blocks, such ashydroxy isophthalic acid. The resulting intermediate is then reactedwith a second cap molecule, preferentially containing a functionalmoiety. An exemplary synthetic route is outlined in the Examplessection.

Attachment at (aa) in FIG. 2

The syntheses of exemplary cap molecules, containing a functional group,are outlined below. Compound 13 can be prepared by following thesynthetic route presented in Scheme 1. Compound 13 can then betransformed into compound 9, using the synthetic approach outlined inExamples 4, 5, 6 and 7.

Multiple functional groups can be attached according to the exemplarydescription provided in the following scheme. Other methods of attachingdifferent functional groups can occur according to methods known to oneof skill in the art.

Attachment at (bb) in FIG. 2

Scheme 3 outlines an exemplary method of synthesizing a compound of theinvention.

Attachment at (cc) in FIG. 2

Scheme 4 outlines an exemplary method of synthesizing compound II.

Scheme 5 outlines an exemplary method of synthesizing compound 10.

Compound 15 can be prepared using the procedure outlined in Scheme 5.Subsequently, 15 can be transformed into compound 10, using thesynthetic steps described in Examples 4, 5, 6 and 7 in addition to asynthetic step useful for the reduction of the nitro group.

Scheme 6 outlines an exemplary method of synthesizing compound IIa.

Scheme 7 outlines an exemplary method of synthesizing compound 11b.

Scheme 8 outlines an exemplary method of synthesizing compound 12.

Compound 16 can be synthesized using the procedure outlined in Scheme 6and can be used as the starting material to synthesize compound 12following the synthetic steps outlined in Examples 4, 5, 6 and 7.

Scheme 9 outlines another exemplary method of synthesizing a compoundthat is similar to compound 16, but uses a different amine protectionscheme.

Attachment at (dd) in FIG. 2

Scheme 10 outlines an exemplary method of synthesizing a compound with afunctional moiety attached to the (dd) position. While this schemedisplays the synthesis of a complexing agent with a functional moiety atboth the (cc) and (dd) positions, a complexing agent with a singlefunctional moiety at the (dd) position (or an additional functionalmoiety at another position) can be synthesized by utilizing one or moreof the syntheses described herein.

Attachment at (ee) in FIG. 2

Scheme 11 outlines an exemplary method of synthesizing a compound with afunctional moiety attached to the (ee) position. While this schemedisplays the synthesis of a complexing agent with a functional moiety atboth the (cc) and (ee) positions, a complexing agent with a singlefunctional moiety at the (ee) position (or an additional functionalmoiety at another position) can be synthesized by utilizing one or moreof the syntheses described herein.

Additional examples for the synthesis of these molecules can be found inthe Examples section.

Once the complexing agent is formed and purified, the metal complex issynthesized by any of a wide range of art-recognized methods, including,for example, by incubating a salt of the ligand with a metal salt, suchas a lanthanide salt (e.g., lanthanide trihalide, lanthanidetriacetate). The reaction of the complexing agent with the metal ion iscarried out either before or after coupling the complexing agent to atargeting moiety in order to generate a complex of the invention.

Attaching a First Cap Moiety

Scheme 12 outlines an exemplary method of synthesizing a cap moleculeattached to a phthalamidyl moiety.

Attaching a Second Cap Moiety

A second cap moiety can be added to a molecule described hereinaccording to Scheme 13.

Another exemplary method of attaching a second cap moiety is describedin Scheme 14.

Another exemplary method of attaching a second cap moiety is describedin Scheme 15.

Another exemplary method of attaching a second cap moiety is describedin Scheme 16.

Another exemplary method of attaching a second cap moiety is describedin Scheme 17.

Functionalizing L¹¹-X

A variety of linker moieties can be attached to the compounds of theinvention. The choice of linker moiety is informed by the moiety towhich the molecule will be attached. For example, the following schemehas a maleimide moiety, which is useful for attachment to thiol moietiessuch as cysteine. Conditions for the attachment of a maleimide moiety toa compound of the invention are shown in Scheme 18.

Another exemplary method for functionalizing L¹¹-X is described inScheme 19.

Another exemplary method for functionalizing L¹¹-X is described inScheme 20. In this scheme, a phosphoramidite derivative of a compound ofthe invention is described for incorporation into an oligonucleotidechain.

Another exemplary method for functionalizing L¹¹-X is described inScheme 21. In this scheme, a carbohydrate-conjugated compound of theinvention is described.

The hydroxy groups in saccharides or polysaccharides can be easilyprotected with acetoxy or benzyl groups. The protected carbohydrates canbe derivatized with carboxyl or amino groups. Typical examples are:

The above-recited synthetic schemes are intended to be exemplary ofcertain embodiments of the invention, those of skill in the art willrecognize that many other synthetic strategies for producing the ligandsof the invention are available without resort to undue experimentation.

The substituents on the isophthalamidyl group and the on the cappingmolecules joining the isophthalamidyl groups can themselves comprisechelating agents other than a hydroxyisophthalamidyl group. Preferably,these chelators comprise a plurality of anionic groups such ascarboxylate or phosphonate groups. In a preferred embodiment, thesenon-PL chelating agents are selected from compounds which themselves arecapable of functioning as lanthanide chelators. In another preferredembodiment, the chelators are aminocarboylates (i.e. EDTA, DTPA, DOTA,NTA, HDTA, etc. and their phosphonate analogs such as DTPP, EDTP, HDTP,NTP, etc).

Many useful chelating groups, crown ethers, cryptands and the like areknown in the art and can be incorporated into the compounds of theinvention. See, for example, Pitt et al., “The Design of ChelatingAgents for the Treatment of Iron Overload,” In, INORGANIC CHEMISTRY INBIOLOGY AND MEDICINE; Martell, Ed.; American Chemical Society,Washington, D.C., 1980, pp. 279-312; Lindoy, THE CHEMISTRY OFMACROCYCLIC LIGAND COMPLEXES; Cambridge University Press, Cambridge,1989; Dugas, BIOORGANIC CHEMISTRY; Springer-Verlag, New York, 1989, andreferences contained therein.

Additionally, a manifold of routes allowing the attachment of chelatingagents, crown ethers and cyclodextrins to other molecules is availableto those of skill in the art. See, for example, Meares et al.,“Properties of In Vivo Chelate-Tagged Proteins and Polypeptides.” In,MODIFICATION OF PROTEINS: FOOD, NUTRITIONAL, AND PHARMACOLOGICALASPECTS;” Feeney, et al., Eds., American Chemical Society, Washington,D.C., 1982, pp. 370-387; Kasina et al., Bioconjugate Chem., 9: 108-117(1998); Song et al., Bioconjugate Chem., 8: 249-255 (1997).

In other embodiments substituents on the isophthalamidyl group or on thebackbone are luminescence sensitizers. Exemplary sensitizers includerhodamine 560, 575 and 590 fluoresceins, 2- or 4-quinolones, 2 or4-coumarins, or derivatives thereof e.g. coumarin 445, 450, 490, 500 and503, 4-trifluoromethylcoumarin (TFC),7-diethyl-amino-cumarin-3-carbohyddzide, etc., and especiallycarbostyril 124 (7-amino-4-methyl-2-quinolone), coumarin 120(7-amino-4-methyl-2-coumarin), coumarin 124(7-amino-4-(trifluoromethyl)-2-coumarin), aminomethyltrimethylpsoralen,napthalene and the like.

Complexes

In a second aspect, the invention provides complexes formed between atleast one metal ion and a compound of the invention. In one exemplaryembodiment, the metal is a member selected from the lanthanide group.Exemplary lanthanides include neodymium (Nd), samarium (Sm), europium(Eu), terbium (Tb), dysprosium (Dy) and ytterbium (Yb), of whicheuropium and terbium are preferred. Other lanthanide ions, such aserbium (Er), lanthanum (La), gadolinium (Gd) and lutetium (Lu) areuseful, but generally less preferred. In another preferred embodiment,the complexes of the invention are luminescent.

After the complexing agent is formed and purified, the metal complex canbe synthesized by any of a wide range of art-recognized methods,including, for example, by incubating a salt of the chelate with alanthanide salt such as the lanthanide trihalide, triacetate, and thelike.

Luminescence Modifying Groups (Donor and Acceptor Moieties)

The luminescent compounds of the invention can be used with a wide rangeof energy donor and acceptor molecules to construct luminescence energytransfer pairs, e.g., fluorescence energy transfer (FET) probes.Fluorophores useful in conjunction with the complexes of the inventionare known to those of skill in the art. See, for example, Cardullo etal., Proc. Natl. Acad. Sci. USA 85: 8790-8794 (1988); Dexter, D. L., J.of Chemical Physics 21: 836-850 (1953); Hochstrasser et al., BiophysicalChemistry 45: 133-141 (1992); Selvin, P., Methods in Enzymology 246:300-334 (1995); Steinberg, I. Ann. Rev. Biochem., 40: 83-114 (1971);Stryer, L. Ann. Rev. Biochem., 47: 819-846 (1978); Wang et al.,Tetrahedron Letters 31: 6493-6496 (1990); Wang et al., Anal. Chem. 67:1197-1203 (1995).

A non-limiting list of exemplary donor or acceptor moieties that can beused in conjunction with the luminescent complexes of the invention, isprovided in Table 1.

TABLE 1 Suitable Moieties Useful as Donors or Acceptors in FET Pairs4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid acridine andderivatives: acridine acridine isothiocyanate5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS)4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonateN-(4-anilino-1-naphthyl)maleimide anthranilamide BODIPY Brilliant Yellowcoumarin and derivatives: coumarin 7-amino-4-methylcoumarin (AMC,Coumarin 120) 7-amino-4-trifluoromethylcouluarin (Coumaran 151) cyaninedyes cyanosine 4′,6-diaminidino-2-phenylindole (DAPI)5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red)7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarindiethylenetriamine pentaacetate4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride)4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL)4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC) eosin andderivatives: eosin eosin isothiocyanate erythrosin and derivatives:erythrosin B erythrosin isothiocyanate ethidium fluorescein andderivatives: 5-carboxyfluorescein (FAM)5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF)2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE) fluoresceinfluorescein isothiocyanate QFITC (XRITC) fluorescamine IR144 IR1446Malachite Green isothiocyanate 4-methylumbelliferone orthocresolphthalein nitrotyrosine pararosaniline Phenol Red B-phycoerythrino-phthaldialdehyde pyrene and derivatives: pyrene pyrene butyratesuccinimidyl 1-pyrene butyrate quantum dots Reactive Red 4 (Cibacron ™Brilliant Red 3B-A) rhodamine and derivatives: 6-carboxy-X-rhodamine(ROX) 6-carboxyrhodamine (R6G) lissamine rhodamine B sulfonyl chloriderhodamine (Rhod) rhodamine B rhodamine 123 rhodamine X isothiocyanatesulforhodamine B sulforhodamine 101 sulfonyl chloride derivative ofsulforhodamine 101 (Texas Red) N,N,N′,N′-tetramethyl-6-carboxyrhodamine(TAMRA) tetramethyl rhodamine tetramethyl rhodamine isothiocyanate(TRITC) riboflavin rosolic acid lanthanide chelate derivatives

There is practical guidance available in the literature for selectingappropriate donor-acceptor pairs for particular probes, as exemplifiedby the following references: Pesce et al., Eds., FLUORESCENCESPECTROSCOPY (Marcel Dekker, New York, 1971); White et al., FLUORESCENCEANALYSIS: A PRACTICAL APPROACH (Marcel Dekker, New York, 1970). Theliterature also includes references providing exhaustive lists ofluminescent and chromogenic molecules and their relevant opticalproperties, for choosing reporter-quencher pairs (see, for example,Berlman, HANDBOOK OF FLUORESCENCE SPECTRA OF AROMATIC MOLECULES, 2ndEdition (Academic Press, New York, 1971); Griffiths, COLOUR ANDCONSTITUTION OF ORGANIC MOLECULES (Academic Press, New York, 1976);Bishop, Ed., INDICATORS (Pergamon Press, Oxford, 1972); Haugland,HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (Molecular Probes,Eugene, 1992) Pringsheim, FLUORESCENCE AND PHOSPHORESCENCE (IntersciencePublishers, New York, 1949); and the like. Further, there is extensiveguidance in the literature for derivatizing reporter and quenchermolecules for covalent attachment via readily available reactive groupsthat can be added to a molecule.

The diversity and utility of chemistries available for conjugatingfluorophores to other molecules and surfaces is exemplified by theextensive body of literature on preparing nucleic acids derivatized withfluorophores. See, for example, Haugland (supra); Ullman et al., U.S.Pat. No. 3,996,345; Khanna et al., U.S. Pat. No. 4,351,760. Thus, it iswell within the abilities of those of skill in the art to choose anenergy exchange pair for a particular application and to conjugate themembers of this pair to a probe molecule, such as, for example, a smallmolecular bioactive material, nucleic acid, peptide or other polymer.

In a FET pair, it is generally preferred that an absorbance band of theacceptor substantially overlap a luminescence emission band of thedonor. When the donor (fluorophore) is a component of a probe thatutilizes luminescence resonance energy transfer (LRET), the donorluminescent moiety and the quencher (acceptor) of the invention arepreferably selected so that the donor and acceptor moieties exhibitluminescence resonance energy transfer when the donor moiety is excited.One factor to be considered in choosing the fluorophore-quencher pair isthe efficiency of luminescence resonance energy transfer between them.Preferably, the efficiency of LRET between the donor and acceptormoieties is at least 10%, more preferably at least 50% and even morepreferably at least 80%. The efficiency of LRET can easily beempirically tested using the methods both described herein and known inthe art.

The efficiency of LRET between the donor-acceptor pair can also beadjusted by changing ability of the donor and acceptor to dimerize orclosely associate. If the donor and acceptor moieties are known ordetermined to closely associate, an increase or decrease in associationcan be promoted by adjusting the length of a linker moiety, or of theprobe itself, between the two luminescent entities. The ability of adonor and an acceptor in a pair to associate can be increased ordecreased by tuning the hydrophobic or ionic interactions, or the stericrepulsions in the probe construct. Thus, intramolecular interactionsresponsible for the association of the donor-acceptor pair can beenhanced or attenuated. Thus, for example, the association between thedonor-acceptor pair can be increased by, for example, utilizing a donorbearing an overall negative charge and an acceptor with an overallpositive charge.

In addition to fluorophores that are attached directly to a probe, thefluorophores can also be attached by indirect means. In someembodiments, a ligand molecule (e.g., biotin) is preferably covalentlybound to the probe species. The ligand then binds to another molecule(e.g., streptavidin), which is either inherently detectable orcovalently bound to a signal system, such as a luminescent compound ofthe invention, or an enzyme that produces a luminescent compound byconversion of a non-luminescent compound. Useful enzymes of interest aslabels include, for example, hydrolases, particularly phosphatases,esterases and glycosidases, or oxidoreductases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.,as discussed above. For a review of various labeling or signal producingsystems that can be used, see, U.S. Pat. No. 4,391,904.

Means of detecting luminescent labels are well known to those of skillin the art. Thus, for example, luminescent labels can be detected byexciting the fluorophore with the appropriate wavelength of light anddetecting the resulting luminescence. The luminescence can be detectedvisually, by means of photographic film, by the use of electronicdetectors such as charge coupled devices (CCDs) or photomultipliers andthe like. Similarly, enzymatic labels may be detected by providing theappropriate substrates for the enzyme and detecting the resultingreaction product.

Methods

The compounds and complexes of the invention are useful as probes in avariety of biological assay systems and diagnostic applications. Anoverview of assay systems, such as competitive assay formats,immunological assays, microarrays, membrane binding assays and enzymeactivity assays, is given e.g., in U.S. Pat. No. 6,864,103 to Raymond etal., which is incorporated herein in its entirety for all purposes. Itis within the ability of one of skill in the art to select and prepare aprobe that includes a complex of the invention, and which is suitablefor each assay system. In an exemplary embodiment, the luminescent probemolecule is used to detect the presence or absence of an analyte in asample.

Thus, in one aspect, the invention provides a mixture of a complex ofthe invention and an analyte.

In a another aspect, the invention provides a method of detecting thepresence or absence of an analyte in a sample. The method comprises (a)contacting the sample and a composition including a complex of theinvention; (b) exciting the complex; and (c) detecting luminescence fromthe complex. The presence or absence of the analyte can be indicated bythe absence or presence of luminescence from the complex.

In a further aspect, the invention provides a method of detecting thepresence or absence of an analyte in a sample. The method comprises (a)contacting the sample and a composition comprising a complex of theinvention, and a luminescence modifying group, wherein energy can betransferred between the complex and the luminescence modifying groupwhen the complex is excited, and wherein the complex and theluminescence modifying group can be part of the same molecule or be partof different molecules; and (b) exciting said complex; and (c)determining the luminescent property of the sample, wherein the presenceor absence of the analyte is indicated by the luminescent property ofthe sample.

In an exemplary embodiment, the analyte, if present in said sample,competes with a probe molecule that includes a complex of the invention,for binding to a binding site located on a recognition molecule. Inanother exemplary embodiment, the analyte displaces the probe moleculefrom the binding site located on a recognition molecule, by binding tothe binding site. In a further exemplary embodiment, the probe moleculeis a complex of the invention.

Hence, in one aspect, the invention provides a kit including arecognition molecule and a compound or a complex of the invention.Exemplary recognition molecules include biomolecules, such as wholecells, cell-membrane preparations, antibodies, antibody fragments,proteins (e.g., cell-surface receptors, such as G-protein coupledreceptors), protein domains, peptides, nucleic acids, and the like.

Analytes

The compounds, complexes and methods of the invention can be used todetect any analyte or class of analytes in any sample. A sample maycontain e.g., a biological fluid (e.g., blood of a patient) or tissue.Other samples can e.g., include solutions of synthetic molecules orextracts from a plant or microorganism (e.g., for drug screeningefforts). Exemplary analytes are pharmaceutical drugs, drugs of abuse,synthetic small molecules, biological marker compounds, hormones,infectious agents, toxins, antibodies, proteins, lipids, organic andinorganic ions, carbohydrates and the like. (see e.g., U.S. Pat. No.6,864,103 to Raymond et al. for additional examples of analytes).

The following examples are provided to illustrate selected embodimentsof the invention and are not to be construed as limiting its scope.

EXAMPLES Example 1 Methyl 2-methoxy-3-methylbenzoate (E-1)

To a mixture of 3-methyl-salicylic acid (200 g, 1.32 mol), anhydrouspotassium carbonate (500 gram, 3.6 mol) and dry acetone (3.5 L) in a 5liter round bottle flask, dimethylsulfate (DMS, 210 mL, 2.2 mol) wasadded in several portions. After stirring at room temperature overnight,the mixture was heated at reflux, and the reaction was monitored by TLC.In order to complete the etherification, further additions of DMS andK₂CO₃ may be necessary. When TLC indicated the completion of thereaction, the mixture was refluxed 4 more hours to destroy any remainingDMS. The mixture was filtered, and the filtrate was evaporated to removethe solvents. A pale yellow thick oil was obtained as the raw product,yield 215 g (91%). ¹H NMR (500 MHz, CDCl₃, 25° C.) δ: 2.26 (s, 3H, CH₃),3.78 (s, 3H, OCH₃), 3.85 (s, 3H, OCH₃), 6.98 (t, J=7.5, 1H, ArH), 7.27(d, J=7.5, 1H, ArH), 7.58 (d, J=7.5, 1H, ArH); ¹³C NMR (500 MHz, CDCl₃,25° C.) δ: 15.7, 51.8, 61.1, 123.2, 124.3, 128.8, 132.4, 134.8, 158.1,166.6.

2-Methoxy-isophthalic Acid (E-2)

To a solution of compound E-2 (215 g, 1.19 mol) in a mixture of methanol(2 L) and water (0.5 L), potassium hydroxide pellets (100 gram, 1.5 mol)were added with cooling. The mixture was refluxed overnight, and thenevaporated to dryness; the residue was then dissolved in water (0.5 L)and acidified with 6N HCl. 2-Methoxy-3-methylbenzoic acid precipitatedas white crystals; yield 189 grams, 95%. ¹H NMR (500 MHz, CDCl₃, 25° C.)δ: 2.23 (s, 3H, CH₃), 3.71 (s, 3H, OCH₃), 7.06 (t, J=7.5, 1H, ArH), 7.36(d, J=7.5, 1H, ArH), 7.49 (d, J=7.5, 1H, ArH).

To a mixture of 2-methoxy-3-methylbenzoic acid (75 gram, 0.45 mol) andwater (4 L) in a 5 liter flask equipped with a mechanic stirrer and aheating mantle, sodium hydroxide (20 g, 0.5 mol) was added, and themixture turned to be a clear solution. The solution was heated to 75°C., and potassium permanganate (158 g, 1 mol) was added in severalbatches during 6 hrs. The resulting brown slurry was stirred overnight;in the meantime, the temperature of the reaction mixture was kept in therange of 80-85° C.

The oxidation process was monitored by proton NMR (in D₂O—NaOD). If thecharacteristic peak of 3-methyl at 2.06 ppm in NMR was stillrecognizable, several grams more of potassium permanganate may be addedto ensure the completion of the oxidation reaction. The slurry was thenfiltered to remove the large amount of MnO₂ and the filtrate wasacidified with conc. HCl. It is noted that the precipitation of thecrystalline product is slow. Pure product was obtained as snow-whitecrystals and was collected by filtration yield 75 grams, 85%. ¹H NMR(500 MHz, DMSO-d₆, 25° C.) δ: 3.79 (s, 3H, CH₃), 7.24 (t, J=7.5, 1H,ArH), 7.79 (d, J=7.5, 2H, ArH).

2-Methoxy-isophthalic acid chloride (E-3)

To a solution of 2-methyoxyisophthalic acid E-2 (75 g, 0.41 mol) in drydioxane, thionyl chloride (119 g, 1 mol) and a drop of DMF were addedwith stirring. The mixture was refluxed overnight under N₂, then all thevolatiles were removed by reduced pressure distillation, the residue wasdried under vacuum (0.1 mm Hg) for at least 8 h. This moisture sensitivecompound is pure enough for the next reaction step.

2-Methoxy-bis(2-mercaptothiazolide)isophthalamide (E-4)

To the ice cooled solution of 2-mercaptothiozaline (107 g, 0.9 mol) and150 mL of triethylamine in 350 mL dry THF was added a solution ofcompound E-3 (made from 75 g of 2-methoxy-isophthalic acid) in 300 mLdry THF drop-wise with stirring. A thick yellow slurry was producedwhich was stirred overnight and then filtered. The yellow filter cakewas washed thoroughly with water, air dried and re-crystallized from2-propanol to give 106.7 g of pure product. The filtrate was evaporatedto dryness, dissolved in CH₂Cl₂, extracted with 1N HCl and 1N KOHsuccessively, then purified by flash chromatography to give additional31 g of product, total yield 137.7 g, 84%. ¹H NMR (500 MHz, CDCl₃, 25°C.) (FIG. 7) δ: 3.419 (t, J=7.5, 2H, CH₂), 3.897 (s, 3H, OCH₃), 4.589(t, J=7.5, 2H, CH₂), 7.137 (t, J=7.5, 1H, ArH), 7.433 (d, J=7.5, 1H,ArH). ¹³C NMR (500 MHz, CDCl₃, 25° C.) δ: 29.2, 55.6, 62.9, 123.1,128.1, 131.9, 154.7, 167.1, 200.8. Anal. Calcd (Found) forC₁₅H₁₄N₂O₃S₄.H₂O (352.427): C, 43.25 (43.02); H, 3.87 (3.78): N, 6.72(6.81).

Additional methods for functionalizing the isophthalamidyl moiety aredescribed herein concerning functionalization at the (dd) and (ee)positions of FIG. 2.

Example 2 H(2,2)-amine or PENTEN (E-5)

Compound E-5 was synthesized by a slight modification of the reportedprocedure (Bianke K. Wagnont and Susan C. Jackels, Inorg. Chem. 1989,28, 1923-1927):

ClEtNHTs (E-5B)

2-Chloroethylamine hydrochloride (E-5A) 70% aqueous solution (1 mol) andK₂CO₃ (1.2 mol) were dissolved in distilled water (4 Liter). TsCl (1mol) was added slowly with stirring. The reaction mixture was stirred atroom temperature for about 24 h. The pH of the reaction mixture wasadjusted to 9 by slow addition of 4 M KOH solution and the mixture keptstirring until TLC indicated all the TsCl were quenched. The resultingprecipitate was collected by using suction filtration, washed withdistilled water, and dried in vacuo (220 g, 95% yield). mp: 77-78° C. ¹HNMR (CDCl₃): δ 2.4 (3H, s), 3.28 (2H, q), 3.52 (2H, t), 5.2 (1H, s), 7.4(2H, dd), 7.9 (2H, dd).

Tosylaziridine (E-5C)

ClEtNHTs (100 g, 0.4 mol) was added to a stirred solution of NaOH (1200mL, 1.4 M) in a salt/ice bath, and stirring was continued for about 1.5h. The precipitate was then allowed to settle for overnight at 10° C.The product was collected, washed with cold distilled water, and driedin vacuo (180 g, 92% yield). mp: 51-52° C. ¹H NMR (CDCl₃): δ 2.3 (4H,s), 2.4 (3H, s), 7.3 (2H, dd), 7.8 (2H, dd).

Penten-4-Ts(N,N,N′,N′-tetrakis(tetrakis(2-((p-tolylsulfonyl)amino)-ethyl))ethylenediamine)(E-5D)

Tosylaziridine (80.8 g, 0.41 mol) was dissolved in dry toluene (160 mL)and acetonitrile (80 mL). A solution of ethylenediamine (6 g, 0.1 mol)in acetonitrile (80 mL) was added dropwise over a period of 1 h. Themixture was then heated at 60-65° C. overnight with stirring. Aftercooling, penten-4-Ts was collected as white fine crystals, washed withacetonitrile and vacuum dried at room temperature, yield 90%. ¹H NMR(CDCl₃): δ 2.41 (s, 12H, CH₃), 2.50 (s, 8H, CH₂), 2.95 (s, 12H, CH₂),5.95 (br, 4H, NHTs), 7.2-7.9 (m, 16H, aromH).

Penten.6HBr (E-5E)

Compound E-5D (42.5 g, 0.5 mol) was dissolved in a mixture of HBr (300mL) and acetic acid (200 mL) in a 1 L round-bottom flask. The flask wasfitted with a condenser and heated to reflux for 48 h and then placed inan ice bath. The resulting precipitate was collected, washed withmethanol and dried in vacuo (36 g, 95% yield). ¹H NMR (D₂O): δ 2.53 (8H,d), 2.60 (2H, d), 2.63 (2H, d), 2.66 (8H, m), 4.1 (8H, s).

Penten (E-5)

Penten (E-5) was prepared from the hydrobromide salt by ion-exchangechromatography. Dowex 1×8 resin in the basic (OH⁻) form was regeneratedby using a 1% NaOH wash, followed by washes with CO₂-free waterpenten.6HBr (4.0 g, 0.4 mol) in CO₂-free water (40 mL) was loaded on theregenerated Dowex resin column (100 mL bed volume). The column waseluted with water, and fractions testing basic were collected. Thecollected fractions were evaporated to produce an oil (1.2 g, 95%yield). ¹H NMR (D₂O): d 2.05 (s, 4H), 2.16 (t, 8H), 2.45 (t, 8H), 4.15(s, 8H).

Example 3 Synthesis of functionalized H(2,2)amine cap (E-4)

(5-Benzyloxycarbonylamino-6-hydroxy-hexyl)-carbamic acid tert-butylester (Cbz-Lys(Boc)-alcohol) (E-8)

This compound was synthesized in 2001 in a 69% yield. Ripka, et al.,Org. Lett., 2001, 3(15), 2309. We found the routinely used mixedanhydride method (G. Kototos, Synthesis, 1990, 299) did not provide pureproduct, but the general CDI procedure (Kim et al., Synlett, 1999, 1239)can provide pure product with satisfactory yield. This procedure wasmodified slightly as follows:

To a stirred solution of2-benzyloxycarbonylamino-6-tert-butoxycarbonylamino-hexanoic acid(Cbz-Lys(Boc)-OH) (Chem-Impex International, 3.8 g, 10 mmol) in THF (25mL) in a 100 mL round flask was added 1,1′-carbonyldiimidazole (CDI)(1.7 g, 10.5 mmol) at room temperature. After 20 minutes, the THFsolution was transferred via a Teflon cannula (+=2 mm) to a stirredsolution of sodium borohydride (0.75 g, 20 mmol) in water (10 mL) in a 1L round flask immersed in a water bath at 5-10° C. The addition caused astrong evolution of hydrogen gas and the mixture was stirred for acouple of hours. The volatiles were then removed on a rotovap, and theresidue was dissolved in ethyl acetate (150 mL). The ethyl acetatesolution was extracted successively with cold 1 N HCl (2×50 mL),saturated sodium bicarbonate solution (2×50 mL), brine (100 mL), and wasdried with anhydrous sodium sulfate. The dried ethyl acetate solutionwas then pass through a one inch flash pad of silica gel, andconcentrated to provide a colorless solid (3.3 g, 91%), TLC R_(f)=0.24(95:5:2 EtOAc:MeOH:H₂O).

¹H NMR (500 MHz, CDCl₃, 25° C.): δ 1.34-1.59 (m, 6H, Lys CH₂), 1.42 (s,9H, Boc CH₃), 2.29 (s, br, 1H, CH₂OH), 3.11 (m, 2H, CH₂), 3.63 (m, 3H,CH+CH₂), 4.60 (s, 1H, BocNH), 5.08 (s, 2H, CH₃), 5.10 (s, 1H, CbzNH),7.32 (m, 5H, ArH). ¹³C NMR (500 MHz, CDCl₃): δ, 22.66, 28.41, 29.89,39.73, 52.98, 64.82, 66.77, 79.28, 128.09, 128.12, 128.52, 136.47,156.42, 156.79. MS (FAB, NBA) C₁₉H₃₀N₂O₅: [M+H]⁺ 367.2.

(5-Benzyloxycarbonylamino-6-oxo-hexyl)-carbamic acid tert-butyl ester(Cbz-Lys(Boc)-aldehyde) (E-9)

This aldehyde was synthesized in 1990 (McConnell, et al., J. Med. Chem.1990, 33, 86-93) by treating Cbz-Lys(Boc)-methyl ester withdiisobutylaluminum hydride. (Rich et al., J. Org. Chem., 1978, 43,3624). In our laboratory, the corresponding alcohol was converted to thecorresponding amino aldehyde via oxammonium oxidation (Leanna, et al.,Tet. Lett. 1992, 32(35), 5029).

A 500 mL 3-necked Morton flask containing Cbz-Lys(Boc)-alcohol (3.66 g.0.01 mol), TEMPO free radical (0.014 g. 0.0001 mol), and NaBr (1.1 g.0.011 mol) in a bi-phasic mixture of toluene (30 mL)/ethyl acetate (30mL) and water (5 mL) was immersed in a 0° C. ice water bath. With rapidmechanical stirring (1200 rpm), an aqueous solution made from mixing 6%commercial bleach (Cholorox^(ultra)) (14 mL), water (20 mL) and KHCO₃(2.5 g, 0.025 mol) was added through a Teflon tube with a glasscapillary tip over a period of 1 h and stirred for an additional 10 min.The aqueous layer was separated and washed with toluene (10 mL). Thecombined organic layers were washed with a solution of KI (0.1 g)dissolved in 10% aqueous KHSO₄ (15 mL). The iodine-colored organic layerwas then washed successively with 10% aqueous sodium thiosulfate (10mL), pH 7 phosphate buffer (0.2 M, 20 mL) and saturated brine. Dryingwith anhydrous Na₂SO₄, filtration and concentration gave 3.1 g (85%) ofraw aldehyde (E-9) as colorless thick oil, which was purified by flashchromatography (5-20% EtOAc in CH₂Cl₂). The appropriate fractions werecombined and the solvents were removed under vacuum, to leave whitesolid as product. The pure compound retained reactivity if stored in afreezer over months.

¹H NMR (500 MHz, CDCl₃, 25° C.): δ 1.32-1.86 (m, 6H, Lys CH₂), 1.41 (s,9H, Boc CH₃), 3.10 (s, br, 2H, CH₂), 4.27 (m, 1H, CH), 4.58 (s, br, 1H,BocNH), 5.10 (s, 2H, CH₃), 5.53 (d, 1H, J=6 Hz, CbzNH), 7.31 (m, 5H,ArH), 9.57 (s, 1H, aldehydeH). MS (FAB, NBA) C₁₉H₂₈N₂O₅: [M+H]⁺ 365.2.

{2-[(2-Amino-ethyl)-(2-benzyloxycarbonylamino-ethyl)-amino]-ethyl}-carbamicacid benzyl ester (BisCbzTREN) (E-10)

This is also a known compound, (Jap. Pat. No. 11302243, Takayanagi,Hisao, Mitsubishi Chemical Industries). The patent reported a multi-stepsynthesis using ethylenediamine as a starting material. We found thatcompound E-10 can be prepared in a one step reaction under mildconditions:

Benzyl Phenyl Carbonate* (Pittelkow, et al., Synthesis, 2002, 2195)(4.56 g, 20 mmol) was added to a stirring solution of TREN (1.46 g, 10mmol) in absolute EtOH (50 mL) while cooling with an ice bath. Thereaction mixture was stirred over night at room temperature. Thevolatiles were removed in vacuo, dissolved in minimum amount of CH₂Cl₂and loaded onto a flash silica column; compound E-10 was separated bygradient chromatography with 3-10% CH₃OH+1% TEA in CH₂Cl₂. The isolatedappropriate fractions were combined and pass through a strong basicalumina plug, and concentrated to afford pure BisZ-TREN as a thickcolorless oil at 75% yield.

¹H NMR (500 MHz, CDCl₃, 25° C.): δ 2.48 (t, 2H, J=5.5 Hz, CH₂), 2.56 (s,br, 4H, CH₂), 2.67 (t, 2H, J=5.5 Hz, CH₂), 3.23 (s, br, 4H, CH₂), 5.06(s, 2H, CH₂), 5.72 (s, 2H, CbzNH), 7.04 (m, 10H, ArH). ¹³C NMR (500 MHz,CDCl₃): δ, 38.91, 39.35, 53.82, 52.69, 66.28, 127.78, 127.82, 136.58,156.70. MS (FAB, NBA) C₂₂H₃₀N₄O₄: [M+H]⁺ 415.

*Carbonic Acid Benzyl Ester Phenyl Ester Benzyl Phenyl Carbonate

Pittelkow, et al., Synthesis, 2002, 2195

This compound is a known compound, but it is not commercially available.It was prepared by following a published procedure. (Rich et al., J.Org. Chem., 1978, 43, 3624). To a mixture of benzyl alcohol (freshlydistilled, 69.2 g, 0.64 mol), pyridine (64 mL) and CH₂Cl₂ (115 mL) in a500 mL 3-necked flask equipped with a condenser, mechanical stirring andan addition funnel was added phenyl chloroformate (100 g, 0.64 mol) overa period of 1 h. The reaction mixture was stirred for an additional 3 h,and H₂O (160 mL) was added. The organic phase was washed with aqueousH₂SO₄ (2 M; 150 mL), dried over anhydrous Na₂SO₄, filtered andconcentrated in vacuo. The crude product was distilled in vacuum127-131° C./0.1 mm Hg to give the desired compound as colorless oil,Yield: 108 g (94%) (literature yield: 79%).

¹H NMR (500 MHz, CDCl₃, 25° C.): δ 5.37 (s, 2H, CH₂), 7.18-7.48 (m, 10H,ArH). MS (FAB): m/z=372.1 (MH⁺).

(5-Benzyloxycarbonylamino-6-{2-[bis-(2-benzyloxycarbonylamino-ethyl)-amino]-ethylamino}-hexyl)-carbamicacid tert-butyl ester (Tris CbzLysBocTREN) (E-11)

BisCbz-TREN (E-10) (4.14 g, 10 mmol) and Cbz-Lys(Boc)-aldehyde (E-9)(3.64 g, 10 mmol) were mixed in THF (50 mL) at room temperature underN₂. The mixture was stirred for 3 hrs with 0.2 g of activated 4 Åmolecular sieves, then sodium triacetoxyborohydride (3.18 g, 15 mmol)was added and the mixture stirred at room temperature under a N₂atmosphere for 24 h. Aqueous 1 N NaOH was added to quench the reactionmixture, and the mixture was extracted with dichloromethane (3×50 mL).The dichloromethane extract was loaded onto a flash silica gel column.The appropriate fractions of a gradient elution (3-10% methanol indichloromethane) were collected and evaporated to dryness to give a palebeige thick oil, which solidified upon standing, yield: 6.27 g, 82%.

¹H NMR (500 MHz, CDCl₃, 25° C.): δ 1.17 (s, br, 2H, Lys CH₂), 1.24-1.89(m, 4H, Lys CH₂), 1.41 (s, 9H, Boc CH₃), 2.53 (s, br, 8H, Tren CH₂),3.03 (s, 2H, BocNHCH₂), 3.14 (s, 2H, CbzNHCH₂), 3.20 (s, 2H, CbzNHCH₂),3.64 (s, br, 1H, Lysine chiral CH), 4.54 (s, 1H, BocNH), 5.05 (m, 6H,CbzCH₂), 7.27 (m, 15H, ArH). ¹³C NMR (500 MHz, CDCl₃): δ, 22.80, 28.33,29.54, 32.52, 39.40, 39.97, 47.41, 50.85, 53.77, 54.34, 66.45, 78.93,79.27, 127.92, 128.06, 128.35, 136.59, 156.01, 156.68, 156.86. MS (FAB,NBA) C₄₁H₅₈N₆O₈: [M+H]⁺ 763.5.

[5-Benzyloxycarbonylamino-6-((2-benzyloxycarbonylamino-ethyl)-{2-[bis-(2-benzyloxycarbonylamino-ethyl)-amino]-ethyl}-amino)-hexyl]-carbamicacid tert-butyl ester (E-13)

Tris CbzLysBocTREN (E-11) (3.81 g, 5 mmol) and Cbz-aziridine*(E-12)(1.24 g, 7 mmol) were mixed in tert-butanol (50 mL) at room temperatureunder N₂. The mixture was stirred under a N₂ atmosphere at 80° C. for 16hrs until TLC indicated that the reaction had reached completion. Thevolatiles were removed under vacuum and the residue was dissolved indichloromethane. The appropriate fractions of a gradient flash silicagel column (1-7% methanol in dichloromethane) were collected andevaporated to dryness to give a pale beige thick oil; yield: 3.98 g,84.7%.

¹H NMR (500 MHz, CDCl₃, 25° C.): δ 1.24-1.87 (m, 6H, Lys CH₂), 1.42 (s,9H, Boc CH₃), 2.26 (s, br, 2H, CH₂), 2.47 (m, br, 6H, CH₂), 2.58 (s, br,2H, CH₂), 2.93-3.35 (m, br, 8H, CH₂), 5.05 (m, 8H, CbzCH₂), 7.27 (m,20H, ArH).

¹H NMR (300 MHz, DMSO-d₆, 25° C.): δ 1.12-1.55 (m, 6H, Lys CH₂), 1.34(s, 9H, Boc CH₃), 2.60 (m, 2H, CH₂), 2.43 (s, 8H, CH₂), 2.85 (m, 2H,CH₂), 3.01 (s, 6H, CH₂), 3.43 (s, 1H, CH), 4.98 (s, 8H, CbzCH₂), 6.71(t, 1H, J=8 Hz, Amide H), 6.92 (d, 1H, J=8 Hz, Amide H), 7.01 (t, 1H,J=8 Hz, Amide H), 7.07 (t, 2H, J=8 Hz, Amide H), 7.31 (s, br, 20H, ArH).¹³C NMR (500 MHz, CDCl₃): δ, 22.78, 28.37, 29.65, 31.15, 32.96, 38.54,38.67, 40.09, 49.36, 52.09, 52.31, 59.35, 66.47, 69.10, 78.94, 127.94,127.97, 128.02, 128.11, 128.36, 128.38, 136.57, 136.64, 156.01, 156.68.MS (FAB, NBA) C₅₁H₆₉N₇O₁₀: [M+H]⁺ 940.5.

*Aziridine and its CBZ or Boc Derivatives

Aziridine or ethylenimine is a well-known compound. It can be preparedfrom 2-haloethylamine hydrohalides with strong base such as silveroxide; sodium or potassium hydroxide in aqueous solution. Synthesis ofaziridine by treating 2-aminoethyl hydrogen sulfate with sodiumhydroxide was recommended by Organic Synthesis (Allen, et al., “OrganicSynthesis”, V. 30, John Wiley and Son, Inc., New York, N.Y., 1950, pp.38-40) and is the most common preparation method. Due to its hightendency to polymerize, the yield of preparation is low. A yield of 37%of aziridine was reported by Organic Synthesis and is considered a goodyield.

Since aziridine is not commercially available now, the literature method(Reeves et al., J. Amer. Chem. Soc., 1951, 73, 3522) was adopted withslight modifications to synthesize this compound. The key issue of thesynthesis is to generate and vaporize the aziridine instantaneously anddistill rapidly to reduce undesirable polymerization.

A 5-Liter, 3-neck flask fitted with a giant magnetic stir bar, a 250 mLdropping funnel and a prolonged condenser (composed of three condensers)arranged for distillation with a heating mantle was set in awell-ventilated hood (FIG. 5). 100 mL of 14% sodium hydroxide solutionwas placed in the 5-Liter flask, and was heated in a metal heatercontrolled by a regulator. The solution was heated at full capacityuntil the distillation was proceeding at a rapid rate. A cool solutionmade from 63 g 2-aminoethyl hydrogen sulfate, 78 g of sodium hydroxideand 270 mL water was added to the distillation flask through thedropping funnel at a rate such that the amount of liquid in the flaskremained about constant. The superheated distillate that came over at100 to 115° C. was collected in a receiving flask and which was immersedin an ice-bath. The flask has a side arm connected to an amine gas trapfilled with dilute sulfuric acid.

In the literature, the distillate was treated with a huge amount ofsodium hydroxide to salt out the raw aziridine, and it was redistilledto ensure the purity of the product. A large quantity of toxic, stronglybasic waste would be generated and the re-distillation of the highlytoxic and volatile aziridine is not advisable. Because the boiling pointof aziridine and its dimer, the major contaminant, are 56-58° and126-127.5° C. respectively, it is possible to control the purity ofaziridine by only collecting the distillate that boils at 50-115° C. Thedistilled dilute aziridine solution was used directly for preparingBoc-aziridine and CBZ-aziridine without further treatment. The yield ofaziridine was estimated around 60%. For characterization, a fraction ofthe distillate was saturated with excess sodium hydroxide, and theaziridine was separated as a thick oil. Its purity was confirmed by NMRspectroscopy.

¹H NMR (500 MHz, CDCl₃, 25° C.): δ, 0.56 (s, br, 1H, NH), 1.56 (s, 4H,CH₂). ¹³C NMR (500 MHz, CDCl₃): δ, 18.03.

Aziridine-1-carboxylic acid benzyl ester (Cbz-aziridine E-12)

The aziridine concentrations in the distillate calibrated by acid-basetitration were in the range of 0.1 to 0.2 M. The solutions were useddirectly for preparation of Cbz-aziridine and Boc-aziridine.

To a stirred, distilled aziridine solution (500 m, 0.1 mol) in around-bottom flask cooling with an ice bath, three equivalents ofpotassium carbonate was added. After all of the solid dissolved, benzylchloroformate (1.5 equivalents) in ethyl ether (150 mL) was added over 2h. The solution was stirred overnight and warmed to room temperature,and the aqueous phase was extracted with methylene chloride (5×30 mL).The organic phases were combined and passed through a flash silica gelpad and concentrated to afford the Cbz-aziridine as colorless thick oil,yield: 7.2 g, 81%.

¹H NMR (500 MHz, CDCl₃, 25° C.): δ 2.22 (s, 4H, CH₂), 5.14 (s, 2H, CH₂),7.34 (m, 5H, ArH). ¹³C NMR (500 MHz, CDCl₃, 25° C.): δ, 20.51, 65.93,127.3, 127.4, 128.7, 140.9, 159 4.

[5-Amino-6-((2-amino-ethyl)-{2-[bis-(2-amino-ethyl)-amino]-ethyl}-amino)-hexyl]-carbamicacid tert-butyl ester (BocLys-H(2,2)amine) (E-14)

To a glass hydrogenation container with 200 mg of wet 5% Pd/C catalyst,5 mL of methanol was carefully added along the glass wall to cover thecatalyst (Caution: the catalyst is ignitable in the air). A solution oftetraCbzBocLys-H(2,2)amine (E-13) (0.94 g, 1 mmol) in methanol (30 mL)was added to the container. The container was put in a Parr bomb andhydrogenated at 500 psi pressure overnight. TLC showed no startingmaterial remained, and the solvent was removed in vacuo. TheBocLys-H(2,2)amine was obtained in its carbonate form as a clearcolorless thick oil. The raw yield was 90%.

¹H NMR (500 MHz, CDCl₃, 25° C.): δ 1.24-1.87 (m, 6H, Lys CH₂), 1.42 (s,9H, Boc CH₃), 2.41-2.82 (m, br, 10H, CH₂), 2.82-3.23 (m, br, 9H, CH₂),3.29 (s, br, 1H, CH), 5.48 (s, 1H, BocNH), 8.52 (s, 3H, CarbonateNH).¹³C NMR (500 MHz, CDCl₃): δ, 22.63, 28.39, 29.47, 31.11, 37.00, 39.93,49.79, 51.99, 68.79, 78.62, 156.19, 168.99. MS (FAB, NBA) C₅₁H₆₉N₇O₁₀:[M+H]⁺ 404.3722.

It was proved that this raw amine can not be used directly forsuccessful cyclization reaction that leads to a macrotricycle IAMligand. This raw product was passed though a Dowex 1×8 strong basicanion exchange resin to remove the carbonate. The resulted free aminewas used for the next step, the cyclization reaction.

Example 4 Me4H(2,2)IAM-tetrathiazolide (E-6)

To a solution of E-4 (100 g, 0.25 mol) in CH₂Cl₂ (3 L), a solution ofcompound E-5 (1 g, 4 mmol) in 300 mL of CHCl₃ was added drop-wise over aperiod of 24 h. The reaction mixture was applied onto a flash silica gelcolumn packed with CH₂Cl₂ and eluted with 3-5% 2-propanol in CH₂Cl₂ toseparate the unreacted 1. The appropriate fractions of the successivegradient elution (5-20% iso-propanol in CH₂Cl₂) were combined andevaporated to dryness to give pure compound 6 as a yellow foam. Yield4.5 g (83%).

¹H NMR (500 MHz, CDCl₃, 25° C.): δ 2.70 (s, 4H, CH₂), 2.76 (t, J=6.2 Hz,8H, CH₂), 3.43 (t, J=7.2 Hz, 8H, CH₂), 3.53 (q, 8H, J=6.0 Hz, CH₂), 3.85(s, 12H, OCH₃), 4.64 (t, J=7.5, 8H, CH₂), 7.17 (t, J=8.2 Hz, 4H, ArH),7.79 (t, J=5.4 Hz, 4H, ArH), 8.63 (d, J=7.5, 1H, ArH). ¹³C NMR (500 MHz,CDCl₃, 25° C.) δ: 29.2, 37.9, 50.6, 53.5, 55.7, 63.1, 124.3, 127.2,129.1, 132.0, 133.9, 155.6, 164.9, 167.3, 201.4. Anal. Calcd (Found) forC₅₉H₆₄N₁₀O₁₂S₈.H₂O (1367.73): C, 50.93 (51.02); H, 4.86 (4.98): N, 10.24(10.01).

Example 5

Me4BocLysBH(2,2)IAM (E-15)

This pendant trimacrocycle E-15 was synthesized under high dilutionconditions. The free amine (E-6) (1.2 g, 3 mmol), 5 mL of triethylamineand 10 mL of iso-propanol were mixed to form a homogeneous solution anddissolved in 950 mL of chloroform in a 1 L round bottom flask. On theother hand, H(2,2)IAMtetrathiazolide (E-14) (4.05 g, 3 mmol) wasdissolved in 950 mL of chloroform in a separate 1 L round bottom flask.The solutions of the E-6 and E-14 (c.a. 3-4 mM) were addedsimultaneously via a homemade “Teflon tube-glass capillary slow additionsystem” to a 12 L three-neck round flask containing 8 liter ofdichloromethane and 2 mL of triethylamine. The addition rates wereadjusted to about 100-120 mL per 24 h for each reactant for 8-10 days,yielding a pale yellow color in the main reaction flask. It is necessaryto keep the high dilution condition in order to minimize polymericby-products. After all the reactants were consumed, the reaction mixturewas stirred an additional 8 hours. TLC reveals the reaction mixture is acomplicated mixture. Since there are many possible isomers coexistingfor this asymmetric molecule, these isomers appear as different spots onthe TLC plate. The reaction mixture was then passed through a flashsilica plug (200 g) to recycle the solvents; the product and by-productremaining on the plug were washed down with a mixture of 20% isopropanolin CH₂Cl₂ containing 1% triethylamine. To simplify the purification andseparation process, the washing solution containing the macrocycle E-15and other by-products was treated with a basic alumina column then aflash silica gel column, the appropriate fractions of gradient (3-7%MeOH in CH₂Cl₂) elution of the flash silica column were combined,evaporated to provide compound E-15 at 25-30% yield. HPLC reveals thatthe purity of the raw product is about 90%. Further column purificationsare needed to provide pure product.

Two main fractions with very different R_(f) values (0.58 and 0.76,developed with a mixture of AcOH/MeOH/CH₃CN/CH₂Cl₂ in a ratio of0.5/8/10/90) in about equal amount were isolated; Mass spectra revealedthat both fractions are the desired macrocycle (E-15).

¹H NMR (500 MHz, DMSO-d₆, 25° C.): δ 1.24-1.55 (m, 15H, Boc CH₃+LysCH₂), 2.52-2.95 (m, br, 24H, NCH₂), 3.21-3.62 (m, br, 16H, NHCH₂),3.65-3.7 (m, 12H, CH₃), 6.78 (s, 1H, BocNH), 7.01-7.15 (m, 8H, ArH),7.50-7.62 (m, 16H, ArH), 8.15-8.30 (m, br, 8H, amideH). ¹³C NMR (500MHz, CDCl₃): δ 23.12, 28.25, 28.27, 29.76, 31.06, 37.09, 38.36, 46.01,50.12, 52.68, 53.48, 62.53, 62.73, 62.85, 78.73, 124.84, 126.61, 133.42,133.82, 154.89, 155.91, 156.01, 164.69, 165.01, 165.28. MS [(+)—FAB,NBA)] C₆₅H₈₉N₁₃O₁₄: m/Z [M+H]⁺ 1276.7.

LysBH(2,2)IAM (4)

Me₄BocLysBH(2,2)IAM (E-15) (0.22 g, 0.17 mmol) was dissolved in 20 mL ofCH₂Cl₂ in a Schlenk flask with a Teflon stopcock. Under a flow of N₂,the solution was cooled to −10° C. before 1 mL BBr₃ was injected. Theslurry was stirred for 5 days before pumping off the excess BBr₃ andCH₂Cl₂. The remaining light yellow solid was dissolved in methanol (100mL) with cooling. The methanol solution was gently refluxed and leftuncapped to allow the release of volatile boron compounds for 6 hrs. Thesolution was then evaporated to dryness. The residue was dissolved inmethanol (10 mL) and diluted into water (50 mL). The mixed solution wasboiled until the volume reduced to c.a. 10 mL and then was cooled,affording a white solid as product. It was collected by centrifugationand vacuum dried at 40° C. Yield: 150 mg (53%).

Compared to the un-functionalized highly symmetric molecule BCH(2,2)IAM,the NMR of compound 4 is complicated, probably due to the fact that thismolecule is asymmetric and there are several conformers co-existing.

¹H NMR (500 MHz, D₂O—NaOD, 25° C.): δ 0.78-1.25 (m, 6H, LysCH₂),2.15-2.30 (m, 2H, CH₂), 2.40-2.92 (m, 26H, NCH₂), 3.00-3.45 (m, 14H,NHCH₂), 3.66 (s, br, 1H, CH), 6.08-6.52 (m, 4H, ArH), 7.35-7.90 (m, 8H,ArH). MS [(+)—FAB, TG/G)] C₅₆H₇₃N₁₃O₁₂: m/Z 1120.5 [MH⁺]. Anal. Calcd.(Found) for C₅₆H₇₃N₁₃O₁₂.5HBr.8H₂O (1668.95): C, 40.30 (40.29); H, 5.68(5.68); N, 10.91 (10.65).

Example E-6 Syntheses of Macrotricyclic Products Compounds 19, 20, 21

Me₄EtGlutarBocLysBH(2,2)IAM (E-18)

Compound E-15 (0.25 g, 0.2 mmol), was taken up in a 1:1 mixture ofdichloromethane and trifluoroacetic acid (10 mL); the solution wasallowed to stir for 3 hr. After evaporation, the residue was dissolvedin methanol, and the pH of the solution was adjusted to 11 with 0.1 NKOH in methanol solution. This basic solution was loaded onto a basicalumina plug and eluted with 10% methanol in dichloromethane to removethe TFA salt and excess base. After removing the solvent, the de-Bocedmacrocycle E-16 was used directly for next step reaction.

The raw compound E-18 was dissolved in dry DMAA (2 mL) and mixed withexcess (2 equiv.) of ethyl glutarate NHS ester in dry dichloromethanesolution (10 mL). The reaction mixture was stirred at room temperaturefor 4 h; the resulted macrocycle E-18 was purified by gradient flashsilica chromatography (2-7% MeOH in CH₂Cl₂).

¹H NMR (500 MHz, DMSO-d₆, 25° C.): δ 1.14 (t, 3H, CH₃), 1.20-1.60 (m,6H, CH₂), 2.05 (m, 2H, CH₂), 2.26 (m, 2H, CH₂), 2.52-2.80 (m, br, 23H,NCH+NCH₂), 3.21-3.62 (m, br, 16H, NHCH₂), 3.65-3.7 (m, 12H, CH₃), 4.03(m, 2H, CH₂), 5.73 (s, 1H, amideH), 7.01-7.15 (m, 4H, ArH), 7.32 (d, 1H,amideH), 7.50-7.65 (m, 8H, ArH), 7.75 (m, 1H, amideH), 7.95 (m, 1H,amideH), 8.10-8.20 (m, 4H, amideH), 8.26 (m, 1H, amideH). ¹³C NMR (500MHz, CDCl₃): δ 13.7, 20.5, 22.6, 24.8, 28.5, 32.6, 32.9, 34.7, 37.3,38.4, 45.5, 50.8, 52.8, 53.1, 59.8, 62.0, 62.3, 62.6, 124.0, 127.5,133.5, 155.0, 155.1, 164.9, 165.4, 172.0, 172.7. MS [(+)—FAB, NBA]:C₆₇H₉₁N₁₃O₁₅: m/Z 1318.8 [MH⁺].

GlutarLysBH(2,2)IAM (5a)

Compound 18 (0.4 g, 0.3 mmol) was deprotected with BBr₃ as described forcompound 4, the deprotected compound 5a was collected by centrifugationand vacuum dried at 40° C. to yield a beige solid was as product. Yield:150 mg (53%).

¹H NMR (500 MHz, D₂O—NaOD, 25° C.): δ 0.78-2.25 (m, 10H, CH₂), 2.40-2.92(m, 26H, NCH₂), 3.00-3.85 (m, 17H, NHCH₂), 6.08-6.52 (m, 4H, ArH),7.35-7.90 (m, 8H, ArH). Anal. Calcd. (Found) forC₆₁H₇₉N₁₃O₁₅.3HBr.5.5H₂O (1576, 184): C, 46.48 (46.44); H, 5.96 (5.98);N, 11.55 (11.39). MS [(+)—FAB, TG/G] C₆₁H₇₉N₁₃O₁₅: m/Z 1234.5 [MH⁺].

GlutarLysBH(2,2)IAM-NHS ester (6a)

To a solution of GlutarLysBH(2,2)IAM (16 mg, 0.01 mmol) in dry DMF (2mL), excess of NHS (3 equiv.) and a catalytic amount of DMAP (2 mg) wasadded. The solution was stirred for 30 min, and DCC (2 equiv.) wasadded. After the reaction mixture was stirred for 4 h, anotherequivalent amount of DCC was added, the solution was stirred overnightunder nitrogen. The mixture was divided into a 1:1 mixture ofcyclohexane and 2-propanol (5 mL) and stirred for 30 min. thencentrifuged to separate the precipitate from the mother liquor. Theprecipitate was suspended in 5 mL 2-propanol with vigorous stirring towash away any low molecular weight impurities and then centrifuged. Suchwashing-centrifuging process was repeated 3 times and the precipitatewas dried under vacuum. FAB(+) Mass spectrum showed the MH+ ion (1331)without showing the un-activated molecular ion peak (1234). No efforthas been made to further characterize this compound yet. New evaluationmethods for these activated compounds are being developed.

Method of Making the Linker

Example 7 Functionalizing the Macrocyclic Ligand (5)

Compound 5 was synthesized by coupling compound 4 with diglycolicanhydride according to Scheme 23 below.

Example 8 Non-Specific Interaction with Protein Streptavidin

It was previously observed that certain ligands such as 2 exhibitnon-specific interactions with proteins, and the addition of a smallamount of non-ionic detergent such as Tween-20 seemed to stabilize theluminescence of Tb-2 for short periods relative to solutions containingno Tween-20. However, the presence of detergents is undesirable in manyapplications.

It was therefore important to determine the tendency of the novelligands to non-specifically interact with proteins. Sulfo-NHS ester andNHS ester derivatives of compound 5 were synthesized and conjugated tostreptavidin. For comparison, the NHS ester of ligand 2 was alsoconjugated to streptavidin. Streptavidin was equilibrated in carbonatebuffer, pH 9, at a concentration of approximately 130 μM. A DMF solutionof the activated esters of each ligand was added to the protein at afinal concentration of approximately 1 mM. The mixtures were allowed toincubate at 4° C. for 1 h. The metal is added either before or afterconjugation of the ligand to the protein. Non-conjugated ligand wasseparated from conjugated protein using a G50 gel filtration centrifugalspin column technique (Penefsky, H. S, Methods Enzymol 1979, 56:527-30).This technique dilutes protein solutions to a lesser extent thanconventional gel filtration techniques. Consecutive spin columns wererun on a single protein sample solution to evaluate the extent ofnon-specific absorption of ligand. Following a single gel filtrationseparation, most of the excess ligand is removed and left at the top ofthe gel bed. A second spin column treatment should leave no furtherligand at the top of the second column unless non-covalent associationof the ligand with the protein occurs.

Results

A significant amount of ligand 2 was left at the top of the gel bed ofthe second and even third consecutive gel filtration spin columns asvisualized by a high degree of residual luminescence. On the other hand,the residual luminescence for activated 4 (both sulfo-NHS and NHS) wasclose to background. Thus, the separation of unconjugated 4 fromconjugated streptavidin was efficient and non-specific binding of thoseligands to streptavidin was found to be minimal.

These experiments demonstrated that the characteristic non-specificassociation with proteins observed for the acyclic ligand 2 is largelyreduced and almost eliminated with the macrocyclic ligand 4. Theseresults may be explained by the greater susceptibility of the acyclicligands to open, when compared to the macrocycle, which has fewerdegrees of freedom. The more constrained nature of the macrocycle mayrender the hydrophobic moieties less accessible and limit non-specificinteractions of those groups with proteins.

The macrocyclic ligand 4 was conjugated to a variety of proteins otherthan streptavidin including protein A and several antibodies; the amountof non-covalent association of the ligand with these proteins was foundto be insignificant compared to the levels observed for ligand 2.

Example 9 Stability of Terbium (Tb) Complexes Stability of Tb Complexesin the Presence of Acid or EDTA

To evaluate the stability of the macrocyclic complexes, the complexeswere exposed to fairly harsh conditions such as 1% acetic acid and 5 mMEDTA solutions. 10% acetic acid is often used in the fixing and stainingof gels, while EDTA is a commonly used preservative often used at aconcentration of 1 mM.

Results

Studies revealed that Tb-3 complexes (at μM concentrations) exhibitstrong Tb emission in 1% AcOH lasting longer than 1 h, whereas Tb-1complexes lose their characteristic luminescence immediately upondilution with 1% AcOH. Similarly, the luminescence of Tb-3 at μMconcentrations is stable for greater than 1 h in 5 mM EDTA, while Tb-1luminescence dissipates in less than 5 min. The reluctance of ligand 3to release the Tb³⁺ ion in the presence of a large excess of EDTAindicates a much higher kinetic stability at neutral pH (in addition tofairly acidic conditions) as compared to ligand 1. High complexstability is crucial when developing applications requiring the use ofisophthalamide complexes at relatively dilute concentrations in complexaqueous solutions (<1×10⁻⁸ M) where the complex is exposed to a varietyof molecules and materials. Without high stability at high dilution,reproducibility is poor and confidence in quantitative resultsdecreases.

Comparison of Kinetic Stability for Different Tb Complexes in VariousMedia

In order to investigate the stability of macrocyclic ligands further,the stabilities of different Tb-complexes were determined using avariety of different media, including 1% acetic acid and 1 mM EDTA.Stock solutions of Tb complexes of compounds 1, 3 and 5a (1 uM) wereprepared in TBST, 50 mM Tris, 150 mM NaCl, 0.05% Tween-80, pH 7.6. Eachstock solution was then diluted 200× into different test solutions inpolypropylene microcentrifuge tubes to a final concentration of 5 nMTb-complex. Each test solution also contained 0.05% Tween-80. The testsolutions were allowed to incubate at room temperature. Samples weretaken at indicated time points, evaluated in triplicate and luminescencewas recorded using standard time-resolved luminescence settings and 340nm and 490 excitation and emission filters, respectively. Results aresummarized in FIG. 1 (compound 1), FIG. 2 (compound 3) and FIG. 3(compound 5a). The results demonstrate a significant increase instability for terbium complexes derived from macrocyclic ligands (3 and5a) when compared to a comparable, “non-restrained” ligand (1). Theresults further demonstrate that substitution of the macrocyclic ligand(3) can further increase the stability in certain media.

Stability of Tb Complexes During SDS-PAGE

The protein conjugated ligands described in Example 3 were evaluated bySDS-PAGE. All samples were boiled at 95° C. for 5 min after being mixed1:1 with 2× reducing or non-reducing SAB. Bufferless Phastsystem gels(GE Healthcare) were loaded according to the manufacturer'sinstructions.

Experimental Results

Visible Tb³⁺ luminescence was observed for all labeled protein samplesfollowing sample heating to 95° C. and exposure to SDS. These resultsdemonstrate that the Lanthanide complexes of the present invention areunexpectedly stable under the tested conditions. Time-resolved CCD-basedimaging systems would be useful to further assess the signal-to-noisedifferences in the obtained images. The use of a cutoff filter thatremoves the excitation light would improve image quality further.

Example 10

Steady-state emission spectra were recorded to compare the emission ofcompound 5a-Tb when unconjugated and conjugated to various proteins. Theemission spectrum of unconjugated 5a-Tb is comparable to the emissionspectra obtained for different protein conjugates of 5a-Tb (FIG. 4).Protein conjugates of 5a-Tb were prepared with streptavidin (sAv),bovine serum albumin (BSA), bovine gamma globulin (BgG), andimmunoglobulin G (IgG). Briefly, the sulfo-NHS ester of 5a was preparedin situ by mixing 5a, EDC and sulfo-NHS in dry DMF and for one hour. Thesulfo-NHS ester of 5a was then added dropwise to a stirring proteinsolution equilibrated in 100 mM carbonate, pH=9.0 and cooled to 4°.Reactions were allowed to proceed until an average of three to five5a-Tb molecules were covalently bound per protein molecule. Luminescencemeasurements were recorded in Tris-buffered saline, pH 7.6. Excitationwas carried out at 340 nm. Plots are displayed with a layered offset forclarity.

Comparison of the luminescence decay lifetimes of unconjugated andstreptavidin conjugated 5a-Tb show that the luminescence emissionlifetimes are essentially the same (FIG. 5). Measurements were conductedon a Fluorolog-3 from Jovin Yvon incorporating a xenon flash lamp anddecay lifetime detection hardware.

Steady-state absorption and emission spectra were recorded for compound4-Tb (FIG. 6). A broad absorption peak centered at 340 nm ischaracteristic of the sensitizing 2-hydroxyisophthalamide chelatingunits. The emission spectrum was recorded by exciting at 340 nm and ischaracteristic of luminescent terbium complexes with emission peakscentered at 488 nm, 545 nm, 585 nm and 618 nm.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this document and scopeof the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference for allpurposes.

1. A compound having a structure according to Formula I:

wherein said compound is covalently modified with at least onefunctional moiety; each Z is a member independently selected from O andS; L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹ and L¹⁰ are linker groupsindependently selected from substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl and substituted orunsubstituted heterocycloalkyl; A¹, A², A³ and A⁴ are membersindependently selected from the general structure:

wherein each R¹ is a member independently selected from H, anenzymatically labile group, a hydrolytically labile group, ametabolically labile group and a single negative charge; and each R⁵, R⁶and R⁷ is a member independently selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl, halogen,CN, CF₃, acyl, —SO₂NR¹⁷R₁₈, —NR¹⁷R¹⁸, —OR¹⁷, —S(O)₂R¹⁷, —COOR¹⁷,—S(O)₂OR¹⁷, —OC(O)R¹⁷, —C(O)NR¹⁷R¹⁸, —NR¹⁷C(O)R¹⁸, —NR¹⁷SO₂R¹⁸, and NO₂,wherein R⁶ and a member selected from R⁵, R⁷ and combinations thereofare optionally joined to form a ring system which is a member selectedfrom substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl andsubstituted or unsubstituted heteroaryl, R¹⁷ and R¹⁸ are membersindependently selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl and substituted orunsubstituted heterocycloalkyl, and R¹⁷ and R¹⁸, together with the atomsto which they are attached, are optionally joined to form a 5- to7-membered ring.
 2. The compound according to claim 1, having thestructure:

wherein R¹, R², R³ and R⁴ are members independently selected from H, anenzymatically labile group, a hydrolytically labile group, ametabolically labile group and a single negative charge; and R⁵, R⁶, R⁷,R⁸, R⁹ R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are members independentlyselected from H, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, halogen, CN, CF₃, acyl, —SO₂NR¹⁷R¹⁸,—NR¹⁷R¹⁸, —OR¹⁷, —S(O)₂R¹⁷, —COOR¹⁷, —S(O)₂OR¹⁷, —OC(O)R¹⁷,—C(O)NR¹⁷R¹⁸, —NR¹⁷C(O)R¹⁸, —NR¹⁷SO₂R¹⁸, and —NO₂, wherein R¹⁷ and R¹⁸are members independently selected from H, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl andsubstituted or unsubstituted heterocycloalkyl, wherein R¹⁷ and R¹⁸,together with the atoms to which they are attached, are optionallyjoined to form a 5- to 7-membered ring; R⁶ and a member selected fromR⁵, R⁷ and combinations thereof are optionally joined to form a ringsystem; R⁹ and a member selected from R⁸, R¹⁰ and combinations thereofare optionally joined to form a ring system; R¹² and a member selectedfrom R¹¹, R¹³ and combinations thereof are optionally joined to form aring system, wherein said ring system is a member selected fromsubstituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl and substituted orunsubstituted heteroaryl.
 3. The compound according to claim 1, whereinat least one of L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹, L¹⁰, A¹, A², A³ andA⁴ is substituted with a functional moiety.
 4. The compound according toclaim 1, wherein said functional moiety has the structure:

wherein L¹¹ is a linker moiety, which is a member selected fromsubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl and substituted or unsubstituted heteroaryl; and Xis a member selected from a reactive functional group and a targetingmoiety.
 5. The compound according to claim 4, having a structure, whichis a member selected from:


6. The compound according to claim 4, wherein said targeting moietycomprises a member selected from a small-molecule ligand, a peptide, aprotein, an enzyme, an antibody, an antigen, a nucleic acid, acarbohydrate, a lipid and a pharmacologically active molecule.
 7. Thecompound according to claim 4, wherein said targeting moiety issubstituted with a luminescence modifying group allowing luminescenceenergy transfer between said complex and said luminescence modifyinggroup when said complex is excited.
 8. The compound according to claim4, wherein said reactive functional group is a member selected from —OH,—SH, —NH₂, —C(O)NHNH₂ (hydrazide), maleimide, activated ester, aldehyde,ketone, hydroxylamine, imidoester, isocyanate, isothiocyanate,sulfonylchloride, acylhalide and —COOY, wherein Y is a member selectedfrom H, a negative charge and a salt counter-ion.
 9. The compoundaccording to claim 4, wherein X is NH₂ and L¹¹ is a member selected fromsubstituted or unsubstituted C₁ to C₁₀ alkyl and substituted orunsubstituted C₁ to C₁₀ heteroalkyl.
 10. The compound according to claim4, wherein said functional moiety comprises a polyether.
 11. Thecompound according to claim 10, wherein said polyether is a memberselected from polyethylene glycol (PEG) and derivatives thereof.
 12. Thecompound according to claim 11, wherein said polyether has a molecularweight of about 50 to about 10,000 daltons.
 13. The compound accordingto claim 1, wherein said linker moieties L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸,L⁹ and L¹⁰ are members independently selected from substituted orunsubstituted C₁ to C₆ alkyl.
 14. The compound according to claim 13,wherein said linker moieties L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹ and L¹⁰are members independently selected from substituted or unsubstitutedethyl.
 15. A luminescent complex formed between at least one metal ionand a compound according to claim
 1. 16. The complex according to claim15, wherein said metal ion is a lanthanide ion.
 17. The complexaccording to claim 16, wherein said lanthanide is a member selected fromneodynium (Nd), samarium (Sm), europium (Eu), terbium (Tb), dysprosium(Dy) and ytterbium (Yb).
 18. A method of detecting the presence of ananalyte in a sample, said method comprising: (a) contacting said sampleand a composition comprising a luminescent complex according to claim15, (b) exciting said complex; and (c) detecting luminescence from saidcomplex.
 19. A method of detecting the presence of an analyte in asample, said method comprising: (a) contacting said sample and acomposition comprising a luminescent complex according to claim 15 and aluminescence modifying group, wherein energy can be transferred betweensaid luminescent complex and said luminescence modifying group when saidcomplex is excited, and wherein said complex and said luminescencemodifying group can be part of the same molecule or be part of differentmolecules; (b) exciting said complex; and (c) determining theluminescent property of said sample, wherein the presence of saidanalyte results in a change in said luminescent property.
 20. The methodaccording to claim 19, wherein said analyte, if present in said sample,binds to an antibody, wherein said antibody is covalently linked to amember selected from a luminescence modifying group and a complexaccording to claim
 15. 21. The method according to claim 20, whereinsaid analyte is a lipid.